Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules

ABSTRACT

The present application discloses a lentiviral transfer system which includes: (i) a self-inactivating transfer vector comprising: multiple gene units, wherein each gene unit includes a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and (ii) a helper construct which lacks a 5′ LTR, wherein the 5′ LTR has been replaced with a heterologous promoter, in which the helper construct further comprises: a lentiviral env nucleic acid sequence containing a deletion, wherein the deleted env nucleic acid sequence does not produce functional env protein; and a packaging signal contains a deletion, wherein the deleted packaging signal is nonfunctional.

CROSS-REFERENCE TO RELATED APPLICATIONS

The Sequence Listing originally submitted in prior application Ser. No.13/333,882 on Dec. 21, 2011 is incorporated herein by reference. A papercopy of the Sequence listing is submitted electronically herewith.

The present application is a continuation of U.S. application Ser. No.16/687,525 filed on Nov. 18, 2019, which is a divisional application ofU.S. application Ser. No. 13/333,882, filed on Dec. 21, 2011, which is acontinuation of U.S. application Ser. No. 12/581,871 filed Oct. 19,2009, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/243,121, filed Sep. 16, 2009; U.S. ProvisionalApplication No. 61/116,138, filed Nov. 19, 2008; and U.S. ProvisionalApplication No. 61/196,457, filed Oct. 17, 2008, the contents of each ofwhich are incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to the field of medicine, specifically todelivery of multiple therapeutic molecules strategically combined withregulatory elements, using a safe for human use, highly and long-termexpressing in human subject lentiviral gene transfer vector for thetreatment of a condition, disease or disorder.

2. General Background and State of the Art

The present application relates to a gene transfer vector that providesversatility, control, high expression, stable multiple gene expression,tolerability, and safety, for the design of effective gene therapies fordisease. Virus vectors in development each have limitations forresearchers to address.

Identification of target genes involved in neoplastic transformation andtumor progression has encouraged the idea that nucleotide sequences ofcancer-relevant genes could lead to the development of tailoredanticancer agents that lack many of the toxic side effects oftraditional cytotoxic drugs. Mutations of the p53 gene are associatedwith transformation to a malignant phenotype. Transfer of wild-type P53,which plays a critical role in the regulation of cell growth anddownregulation of genes that contribute to cancer progression, is hopedto result in selective and specific inhibition of tumor growth whileminimizing undesirable side effects on normal cells. Delivery of the p53gene has been reported in a replication-deficient adenoviral vectorcontaining the wild-type p53 gene sequence. Functional activity andexpression of the transgene product in tumor cells treated with theadenoviral vector has been reported. (Baker, et al., 1990, Suppressionof human colorectal carcinoma cell growth by wild-type p53, Science 249:912-915.) Two adenovirus-based gene therapeutics for the treatment ofcancer were recently commercialized in China. These two agents, combinedwith chemotherapy, have been used there as an alternative treatment forsome types of refractory cancer. (Peng, 2005, Current Status ofGendicine in China: Recombinant Human Ad-p53 Agent for Treatment ofCancers, Hum. Gene Ther. 16, 1016-1027; Yu, W., and Fang, H., 2007,Clinical Trials with Oncolytic Adenovirus in China. Curr. Cancer DrugTargets 7, 141-148). Although adenovirus vectors can efficiently delivertherapeutic genes in both dividing and non-dividing cells, and can bemanufactured at high viral titers, adenovirus vectors are highlyimmunogenic (Shirakawa, et al., 2008, The Current Status ofAdenovirus-based Cancer Gene Therapy, Mol. Cells 25(4): 462-466).Furthermore, long-term expression in target tissues is not observed.

Lentiviruses, such as HIV, are “slow viruses.” Vectors derived fromlentiviruses can be expressed long-term in the host cells after a fewadministrations to the patients, e.g., via ex vivo transduced bonemarrow stem cells. For most diseases and disorders, including geneticdiseases, cancer, and neurological disease, long-term expression iscrucial to successful treatment. Safety has been a concern withlentiviral vectors, but a number of strategies for eliminating theability of lentiviral vectors to replicate have now been described. Forexample, the deletion of promoter and enhancer elements from the U3region of the long terminal repeat (LTR) are thought to have noLTR-directed transcription. The resulting vectors are called“self-inactivating” (SIN). However, it has been reported thatHIV-1-derived vectors containing the SIN deletion in the U3 region ofthe LTR are capable of expressing full-length genomic transcripts(Logan, et al., 2004, Integrated Self-Inactivating Lentiviral VectorsProduce Full-Length Genomic Transcripts Competent for Encapsidation andIntegration, J. Virology 78(16): 8421-8436). Therefore, combination ofthis deletion with other safety measures must be considered.

The last few years have seen immense excitement regarding the use ofRNAi agents for disease therapies. Investigators reported that they wereable to specifically silence mutant oncogenic ras without affectingwild-type ras in vitro (Zhang, et al., 1995, Safety evaluation ofAd5CMV-p53 in vitro and in vivo, Human Gene Therapy 6:155-164; Ishii, etal., 2001, Potential cancer therapy with the fragile histidine triadgene review of the preclinical studies, JAMA 286: 2441-2449). It isbelieved that treatment costs for siRNA would be similar to mostprotein-based therapies, e.g., antibody therapies. Preclinical cancerstudies have shown inhibition of growth and survival of tumor cells byRNAi-mediated downregulation of several key oncogenes or tumor-promotinggenes, including growth and angiogenic factors or their receptors(vascular endothelial growth factor, epidermal growth factor receptor),human telomerase (hTR, hTERT), viral oncogenes (papillomavirus E6 andE7) or translocated oncogenes (BCR-abl).

Various studies report on the in vivo activity and the potential of RNAiagents to suppress tumor growth. These include an intratumoral injectionof an shRNA-adenoviral vector construct targeting a cell-cycle regulatorcausing inhibition of subcutaneous small cell lung tumor in mice, andsystemic administration of an siRNA targeting a carcinoembryonicantigen-related cell adhesion molecule (CEACAM6) in mice withsubcutaneously xenografted pancreatic adenocarcinoma cells. In anotherreport, direct injection of a plasmid vector expressing shRNAs to matrixmetalloproteinase MMP-9 and a cathepsin showed efficacy in establishedglioblastoma (Chen, et al., 2005, Reversal of the phenotype byK-rasvall2 silencing mediated by adenovirus-delivered siRNA in humanpancreatic cancer cell line Panc-1, World J. Gastroenterol. 11(6):831-838). However, delivery of siRNA for long-term expression in targetcells and tissues has been particularly difficult in vivo.

Another problem in gene transfer is the delivery of therapeuticmolecules to a sufficient number of target cells to elicit a therapeuticresponse. Recently, a series of virus-encoded and other regulatoryproteins were found to possess the ability to cross biologicalmembranes. These proteins include HIV-Tat and the herpes simplex virustype 1 tegument protein VP22. VP22 was also reported to exhibit a uniqueproperty of effecting intercellular spread. VP22 is a basic, 38-kDaphosphorylated protein (Knopf, et al., 1980, J. Gen. Virol. 46:405-414)encoded by the viral UL49 gene (Elliott, et al., 1992, J. Gen. Virol.73:723-726).

Specific and controlled delivery of therapeutic molecules to an affectedcell population, e.g., to tumor cells and even circulating cancer cells,can potentially be achieved by strategically positioning nucleic acidand protein regulatory elements, e.g., cell and tissue-specificpromoters and enzyme cleavage sites. These elements are recognized bythe production machinery that is present only in certain cell types. Theability to easily combine and regulate the expression and delivery ofmultiple therapeutic molecules, while taking effective safety measureswithout compromising expression levels, in methods for using alentiviral gene transfer vector or lentiviral transfer system, wouldprovide a researcher with a critical tool for treating a broad range ofdiseases and disorders.

SUMMARY OF THE INVENTION

The present invention is related to lentiviral transfer systemsincluding safe, self-inactivating, recombinant lentiviral vectors withthe capacity to accommodate strategic combinations of genes fortherapeutic molecules and novel regulatory sequences, in methods fortreating a broad range of diseases and disorders.

In one aspect, the invention relates to a lentiviral gene transfersystem comprising: a self-inactivating transfer vector comprising: afirst gene unit with a first heterologous nucleic acid sequence,operably linked to a first regulatory nucleic acid sequence; and asecond gene unit with a second heterologous nucleic acid sequence,operably linked to a second regulatory nucleic acid sequence; and ahelper construct which lacks a 5′ LTR, wherein said 5′ LTR has beenreplaced with a heterologous promoter, said helper construct furthercomprising: a lentiviral env nucleic acid sequence containing adeletion, wherein said deleted env nucleic acid sequence does notproduce a functional env protein; a packaging signal containing adeletion, wherein said deleted packaging signal is nonfunctional. Thetransfer vector may be preferably derived from HIV-1. Preferably, RNAior a polypeptide may be encoded by the heterologous nucleic acidsequence. In addition, expression of the first and second heterologousnucleic acid sequences may have a synergistic effect in inhibitingprogression of a disease or disorder.

The transfer vector may include mammalian insulator sequence and spliceacceptor and splice donor sites, and may be free of wPRE “wood-chuck”hepatitis virus post-transcriptional element downstream of a cloningsite (Gao et al., J. Virol., Mar. 2008; p. 2938-2951).

The RNAi may inhibit expression of a gene that contributes toprogression of a disease or disorder. In addition, the first or secondheterologous nucleic acid sequence may include a sequence encoding atrafficking signal, and the trafficking signal may be expressed as afusion with a protein expressed from the second heterologus nucleic acidsequence. The intercellular trafficking signal may be amembrane-penetrating protein or a fragment thereof, such as a plant orbacterial protein toxin, or viral protein, any other sequence domainwith transporting function between cells, in particular, cancer cells.The trafficking signal may be derived from herpesvirus VP22 or HIV-Tat,or may be a HIV-Tat eleven amino acid transduction sequence. Theherpesvirus may be HSV1, and the trafficking signal may further be aVP22 protein homologue of HSV1 VP22. The VP22 transport signal mayinclude a C-terminal 34 amino acid sequence of VP22 of HSV1, or afragment having 80% or greater identity to the terminal 34 amino acidsequence of VP22 of HSV1. Further, the VP22 transport signal may includeone or more of RSASR, RTASR, RSRAR, RTRAR, ATATR, or RSAASR.

The transfer vector may utilize general or cell or tissue specificpromoters such as TSTA promoter, mesothelin promoter, hPSA promoter,hCCKAR promoter, hAFP promoter, and hNSE promoter.

Tissue-specific enzyme cleavage sites may be included in the transfervector, wherein cleavage at the site occurs within a polypeptide that isencoded by the first and second heterologous nucleic acid sequences. Theregulatory nucleic acid sequence may include a sequence encoding a cellor tissue-specific enzyme cleavage site, wherein cleavage at the siteoccurs within at least one polypeptide that is encoded by two or more ofthe first, second and third heterologous nucleic acid sequences. Thecell or tissue-specific enzyme cleavage site may be a protease 2Acleavage site, a presecretory protein signal peptidase cleavage site, ora pancreatic prechymotrypsinogen cleavage site.

The transfer vector may also include a nucleic acid sequence encodingtranslation initiation site. Such a sequence may be positioned betweenthe gene units, and in certain aspects may be considered to belong to a“regulatory sequence” of a gene unit. Although a variety of sequencesmay be used, an internal ribosome entry site (IRES) is preferred.

The inventive lentiviral transfer system may be used to prepare atreatment for a disease or disorder. The disease or disorder may becancer. In a preferred embodiment, a heterologous nucleic acid sequencemay encode the P53 protein.

The antisense RNA, RNAi, or any polypeptide expressed from theheterologous nucleic acid sequence may be expressed consistently and forlong period of time to inhibit expression of a gene or the activity of agene product that contributes to progression of the cancer. Theantisense RNA, RNAi and the polypeptide may inhibit expression andactivity of a tumor promoting gene or gene product. The RNAi and theexpressed polypeptide may inhibit expression of or activity of a growthfactor, growth factor receptor, angiogenic factor, angiogenic factorreceptor, cell cycle regulator, apoptosis-inducing molecule, or celladhesion molecule. The RNAi or the expressed polypeptide may inhibit theexpression or activity of a vascular endothelial growth factor, Bc1-2,K-ras, AEC-1, Myc, including c-Myc, a vascular endothelial growth factorreceptor, epidermal growth factor receptor, hTR, hTERT, papillomavirusE6, papillomavirus E7, BCR-abl, CEACAM6, MMP9, or a cathepsin.

The cancer to be treated may be prostate cancer in which a regulatorynucleic acid sequence may include hPSA. If the cancer is liver cancerthen a regulatory nucleic acid sequence may include hAFP. If the canceris pancreatic cancer then a regulatory nucleic acid sequence may includehCCKAR to control the expression of RNAi or a polypeptide.

The inventive lentiviral transfer system may be used to prepare atreatment for a genetic disorder, such as a metabolic disorder,including Gaucher's Disease or Fabry's Disease. In the case of Gaucher'sDisease, a first heterologous nucleic acid sequence may encodeglucocerebrosidase and a second heterologous nucleic acid sequence mayencode a human intrinsic selectable marker such as huCD25 protein orhuNGF protein.

The lentiviral transfer system may include a regulatory nucleic acidsequence that includes a sequence encoding a trafficking signal that isexpressed as a fusion with a glucocerebrosidase protein expressed froman adjacent heterologous nucleic acid sequence. A second regulatorynucleic acid sequence may include translation initiation sequence aswell. A cell or tissue-specific enzyme cleavage site may also beincluded.

In the case of Fabry's Disease, said first heterologous nucleic acidsequence may encode an alpha-galactosidase-A protein, and a secondheterologous nucleic acid sequence may encode the huCD25 protein.

In another aspect, the inventive lentiviral transfer system may includea first heterologous nucleic acid sequence comprising a sequenceencoding a trafficking signal that is expressed as a fusion with thealpha-galactosidase-A protein expressed from the first heterologousnucleic acid sequence. The trafficking signal may be a VP22 traffickingsignal or an HIV-Tat trafficking signal.

In yet another aspect, if the genetic disorder is Leber CongenitalAmaurosis, the first heterologous nucleic acid sequence may encode theRPE65 protein, and a second heterologous nucleic acid sequence mayencode the hBDNF protein, and a second regulatory nucleic acid sequencemay include hNSE, and further the vector may include a thirdheterologous nucleic acid sequence encoding the hNGF protein, and also athird regulatory nucleic acid sequence including a sequence encoding acell or tissue-specific enzyme cleavage site.

In another aspect, the disease or disorder to be treated may be aneurological disorder, such as Alzheimer's Disease, in which case, afirst heterologous nucleic acid may encode the hNGF protein, a secondheterologous nucleic acid may encode an RNAi targeted to beta-amyloidprecursor protein, and wherein the second regulatory nucleic acidsequence may include a sequence encoding a cell or tissue-specificenzyme cleavage site. A third third heterologous nucleic acid thatencodes the hBDNF protein may also be included, in which the firstregulatory nucleic acid sequence may include a sequence encoding atrafficking signal that is expressed as a fusion with the hNGF proteinexpressed from the first heterologous nucleic acid sequence.

If the neurological disorder is Parkinson's Disease, a firstheterologous nucleic acid may encode the hBDNF protein or the hGDNFprotein, and the second heterologous nucleic acid encodes hGAD. Thesecond regulatory nucleic acid sequence may include a sequence encodinga cell or tissue-specific enzyme cleavage site. Further, the firstregulatory nucleic acid may include a sequence encoding a traffickingsignal that is expressed as a fusion with the hBDNF protein or hGDNFprotein expressed from the first heterologous nucleic acid sequence,wherein the vector may further include a third heterologous nucleic acidsequence encoding hNGF, in which the vector may further include a thirdregulatory nucleic acid sequence encoding a cell or tissue-specificenzyme cleavage site.

In another aspect, the invention is directed to a method for treating acondition, comprising administering to a patient a lentiviral particlefor gene transfer, said lentiviral particle produced using a lentiviraltransfer system comprising: a self-inactivating transfer vectorcomprising: a first gene unit with a first heterologous nucleic acidsequence, operably linked to a first regulatory nucleic acid sequence;and a second gene unit with a second heterologous nucleic acid sequence,operably linked to a second regulatory nucleic acid sequence; and ahelper construct which lacks a 5′ LTR, wherein said 5′ LTR has beenreplaced with a heterologous promoter, said helper construct furthercomprising: a lentiviral env nucleic acid sequence containing adeletion, wherein said deleted env nucleic acid sequence does notproduce functional env protein; a packaging signal containing adeletion, wherein said deleted packaging signal is nonfunctional. Thecondition may be cancer, such as liver cancer, pancreatic cancer, orprostate cancer. The condition may also be a genetic disorder, such asGaucher's Disease or Fabry's Disease. The condition may also be aneurological disorder, such as Parkinson's Disease or Alzheimer'sDisease. The condition may also be a need for cosmetic enhancement.

A pharmaceutical composition comprising a lentiviral particle for genetransfer, said lentiviral particle produced using a lentiviral transfersystem comprising: a self-inactivating transfer vector comprising: afirst gene unit with a first heterologous nucleic acid sequence,operably linked to a first regulatory nucleic acid sequence; and asecond gene unit with a second heterologous nucleic acid sequence,operably linked to a second regulatory nucleic acid sequence; and ahelper construct which lacks a 5′ LTR, wherein said 5′ LTR has beenreplaced with a heterologous promoter, said helper construct furthercomprising: a lentiviral env nucleic acid sequence containing adeletion, wherein said deleted env nucleic acid sequence does notproduce functional env protein; a packaging signal containing adeletion, wherein said deleted packaging signal is nonfunctional.

In a further aspect, the invention is directed to a pharmaceuticalcomposition that includes a lentiviral transfer vector, said lentiviraltransfer vector comprising a first heterologous nucleic acid sequence,operably linked to a first regulatory nucleic acid sequence; and asecond heterologous nucleic acid sequence, operably linked to a secondregulatory nucleic acid sequence, wherein said transfer vector isself-inactivating.

The pharmaceutical compositions as described above may further include achemotherapeutic agent, a steroid agent such as prednisolone, cortisone,corticosterone, or dexamethasone.

In one aspect of the invention, SIN element is incorporated into therecombinant lentivirus; the tat region has been modified so as tooptionally allow for infection efficiency without allowing thereplication functions and uncontrolled infection of the recombinantlentivirus beyond the intended target; and the rev protein has beeninactivated so as to prevent further unwanted infectivity whilepreserving the basic function of the rev to support the expressionefficiency of the therapeutic gene(s). Tat and rev proteins may beinactivated of their original replication activity without necessarilyremoving them from the lentivirus and thereby preserve their desirableattributes while keeping the resulting vector bio-safe.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIGS. 1A-1C Lentiviral transfer system. The drawing shows an HIV-1-basedgene transfer system carrying multiple functional (therapeutic) genes.FIG. 1A. Helper (packaging) construct. The 5′ LTR of the helperconstruct is replaced with the CMV promoter, to avoid integration of theviral elements presented in that construct. This is an importantbiosafety feature of the lentiviral transfer system of the invention.The triangles represent deletions: one represents a 36-bp deletionharboring the putative packaging signal from nucleotides 753 to 789between the 5′ major splice donor site and the beginning of the gag ATGcoding region; one represents a deletion in the tat gene, and a thirdrepresents a deletion in nef. The packaging signal is functionallyabsent from this construct, to avoid production of an active gag-polprecursor. The poly (A) site was derived from the bovine growth hormonegene. The helper construct provides a nucleic acid sequence encodinglentiviral gag and pol, operably linked to a heterologous regulatorynucleic acid sequence. The construct further contains a deleted,nonfunctional env protein and is devoid of lentiviral sequences bothupstream and downstream from a splice donor site to a lentiviral gaginitation site. FIG. 1B. Envelope expression construct. An envelopeconstruct encoding vesicular stomatitis virus G glycoprotein (VSV-G) isshown, though other non-lentiviral envelope proteins can be usedinstead. Expression is driven by the HIV-1 LTR. The poly(A) site wasderived from the simian virus 40 late region. FIG. 1C. Transfer vectorconstructs. In these constructs, Tat, Vpr, and Nef are inactivated.Boxes interrupted by jagged lines contain partial deletions.RRE=Rev-response element; ψ=cis-acting packaging signal; IRES=internalribosome entry site; huCD25=human IL-2Ra chain gene; GFP=GreenFluorescent Protein coding sequence; RNAi=interfering RNA codingsequence; S=stop codon; T=termination signal (e.g., SV40 polyA or BGHpolyA); CS=cleavage site (e.g., viral 2A-like peptide cleaved by the 2Aprotease, presecretory protein cleavage site, pancreaticprechymotrypsinogen cleavage site); hPSA=human prostate specific antigenpromoter; P1=promoter 1; CMV, Human CMV-IE promoter; P2=promoter-2;P53=tumor suppressor gene; dsRNA=Bcl-2 RNAi (human).

FIG. 2 Heterologous Proteins. This table provides examples ofheterologous proteins contemplated for use in the vectors and methods ofthe present invention.

FIG. 3 Transport Genes. This table provides examples of transport genescontemplated for use in developing transport sequences for the vectorsand methods of the present invention.

FIG. 4 Promoter Elements. This table provides examples of promoterelements contemplated for use in the vectors and methods of the presentinvention.

FIG. 5 Enhancer Elements. This table provides examples of enhancerelements contemplated for use in the vectors and methods of the presentinvention.

FIG. 6 SIN-LV-P53-EGFP and SIN-LV-BCL2 RNAi-EGFP. The drawing shows thevector constructs used as described in the Examples.

FIG. 7 Therapeutic Constructs. This table provides examples ofconstructs for use in the vectors and methods of the invention forcertain therapeutic applications.

FIGS. 8A-8D Infection of Prostate Cancer Cells. FIG. 8A. EGFP expressionin PC-3 cells infected with STN-HIV-p53-TRES-EGFP (Panel 1) and PC-3cells not infected with virus (2). FIG. 8B. EGFP expression in 293Tcells infected with SIN-HIV-p53-TRES-EGFP (Panel 1) and 293T cells notinfected with virus (2). FIG. 8C. Viral packaging of Vector. FIG. 8D.P53 expression. Lane 1: Size standards 1 kb plus DNA Ladder. Lane 2:PC-3 cells (no infection). Lane 3: PC-3 cells (infection). Lane 4:Blank. Lane 5: 293T cells (no infection). Lane 6: 293T cells(infection).

FIG. 9 Therapeutic Constructs 2. This table provides examples ofconstructs for use in the vectors and methods of the invention forcertain therapeutic applications.

FIGS. 10A-10B Experimental evidence of efficacy of the viral vectorconstruct P53-Bc1-2 RNAi, expressing P53 and an RNAi agent targetinghuman Bcl-2. FIGS. 10A and 10B show phenotype of in vitro cell cultureunder phase-contrast microscope indicating that the P53-Bc1-2 RNAiconstruct induces cell necrosis in PC3 prostate cancer cells. FIG. 10A.untreated living cells. FIG. 10B. treated with viral vector constructP53-Bc1-2 RNAi, expressing P53 and an RNAi agent targeting human Bc1-2.

FIGS. 11A-11B Experimental evidence of efficacy of the viral vectorconstruct P53-Bc1-2 RNAi, expressing P53 and an RNAi agent targetinghuman Bcl-2. FIGS. 11A and 11B show phenotype of in vitro cell cultureunder phase-contrast microscope indicating that the P53-Bc1-2 RNAiconstruct does not cause necrosis in 293T cells. FIG. 11A. untreatedcells. FIG. 11B. treated with viral vector construct P53-Bc1-2 RNAi,expressing P53 and an RNAi agent targeting human Bc1-2.

FIG. 12 In vivo test demonstrates tumor reduction efficacy of viralvector construct P53-Bc1-2 RNAi, expressing P53 and an RNAi agenttargeting human Bcl-2. In order from left to right, far left bar isweight of the control tumor group after three weeks; tumor treated withP53 alone expressed through viral vector construct (repair geneticdefect); tumor treated with BCL2 SiRNA alone expressed through viralvector construct (down regulation of BCL2 gene) only; and on the farright tumor treated with viral vector construct P53-Bc1-2 RNAi,expressing P53 and an RNAi agent targeting human Bcl-2 for simultaneousP53 (repair) and BCL2 siRNA.

FIGS. 13A-13B show in vivo tumor staining of control (untreated tumor)versus treatment groups. Immunohistochemical staining by specificantibodies show that tumor cells treated with viral vector constructP53-Bc1-2 RNAi, expressing P53 and an RNAi agent targeting human Bcl-2no longer produce hPSA (human prostate specific antigen), which meansthat the cells are no longer active tumor cells or are no longeractively cancerous. FIG. 13A. control tumor expresses hPSA. FIG. 13B.treatment tumor shows cell necrosis and no expression of hPSA.

FIGS. 14A-14C show a table that shows results from another mouse (invivo) study confirming tumor reduction findings. Group V1 is prostratetumor mice treated with P53 expressed alone in a viral vector; Group V2is tumor mice treated with BCL2 siRNA alone expressed through viralvector; V3 is tumor mice treated with the dual viral constructexpressing P53 and BCL2 siRNA. Group PC shows untreated tumor micecontrol. “**” in the tables indicates tumor sizes on Day 7 as thestarting sizes for calculating the tumor growth rate and the ratio ofTumor Weigh/Starting Tumor Size for groups of V1, V2, V3, and PC.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

Unless otherwise indicated, all terms used herein have the same ordinarymeaning as they would to one skilled in the art of the presentinvention.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application. All statements as to the date orrepresentations as to the contents of these documents are based on theinformation available to the applicant and do not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

As used herein, reference to “upstream”, “downstream”, “first gene”,“second gene”, “last gene”, “before”, “after” and so forth in relationto the spatial positioning of the various DNA sequences in a vector, ismeant to be with respect to the 5′ to 3′ orientation of the vectorsequence. For example “before” will have the same meaning as “upstreamof” or 5′ of a particular reference position, and “after” will have thesame meaning of “downstream of” or 3′ with respect to a particularreference point on the vector.

As used herein, “gene unit” includes a regulatory region that mayinclude a promoter and a heterologous nucleic acid sequence encodingeither an antisense RNA, RNAi or polypeptide of interest that iscontrolled by the regulatory sequence, which is typically referred to inthe context of a multigene transfer vector. However, in situations wherea fused polypeptide is desirous of being generated from the multigenevector of the encoded polypeptides of adjacent gene units, theregulatory region of the downstream gene units may include a nucleicacid sequence encoding a cleavage site instead of a separate promoter.

MODES OF CARRYING OUT THE INVENTION

It is to be understood that this invention is not limited to particularformulations or process parameters, as these may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting. Further, it is understood that a numberof methods and materials similar or equivalent to those described hereincan be used in the practice of the present invention.

I. Lentiviral Transfer System

The present invention provides a recombinant lentivirus capable ofinfecting dividing and non-dividing cells. The virus is useful for thein vivo and ex vivo transfer and expression of nucleic acid sequences.Lentiviral vectors of the invention may be lentiviral transfer plasmidsor infectious lentiviral particles. Construction of lentiviral vectors,helper constructs, envelope constructs, etc., for use in lentiviraltransfer systems has been described, e.g., in U.S. Patent App. Pub. No.2003/0119770, “Intercellular delivery of a herpes simplex virus VP22fusion protein from cells infected with lentiviral vectors,”incorporated herein by reference in its entirety.

Lentiviruses

Lentiviruses are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a lentivirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. This integrated DNAintermediate is referred to as a provirus. Transcription of the provirusand assembly into infectious virus occurs in the presence of anappropriate helper virus or in a cell line containing appropriatesequences enabling encapsidation without coincident production of acontaminating helper virus. As described below, a helper virus is notrequired for the production of the recombinant lentivirus of the presentinvention, since the sequences for encapsidation are provided byco-transfection with appropriate vectors.

The lentiviral genome and the proviral DNA have three genes: the gag,the pol, and the env, which are flanked by two long terminal repeat(LTR) sequences. The gag gene encodes the internal structural (matrix,capsid, and nucleocapsid) proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase) and the env geneencodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve topromote transcription and polyadenylation of the virion RNAs. The LTRcontains all other cis-acting sequences necessary for viral replication.Lentiviruses have additional genes including vit, vpr, tat, rev, vpu,nef, and vpx (in HIV-1, HIV-2 and/or SIV).

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site, ψ). If thesequences necessary for encapsidation (or packaging of lentiviral RNAinto infectious virions) are missing from the viral genome, the resultis a cis defect which prevents encapsidation of genomic RNA. Theresulting mutant is still capable of directing the synthesis of allvirion proteins, but lacks function of replication.

In a first embodiment, the invention provides a recombinant lentiviruscapable of infecting a dividing or non-dividing cell. The recombinantlentivirus comprises a nucleic acid sequence containing a lentiviralpackaging signal flanked by lentiviral cis-acting nucleic acid sequencesnecessary for reverse transcription and integration, a heterologousnucleic acid sequence operably linked to a regulatory nucleic acidsequence, and a nucleic acid sequence encoding an intercellulartrafficking signal, where the nucleic acid sequence encoding theintercellular trafficking signal is fused in-frame with the heterologousnucleic acid sequence, where the lentivirus does not contain either acomplete gag, pol, tat, rev, or env gene.

The recombinant lentivirus of the invention is therefore geneticallymodified in such a way that some of the structural, infectious genes ofthe native virus have been removed, and some removed sequences replacedwith a nucleic acid sequence to be delivered to a target non-dividingcell. After infection of a cell by the virus, the virus releases itsnucleic acid into the cell and the lentivirus genetic material canintegrate into the host cell genome. The transferred lentivirus geneticmaterial is then transcribed and translated, e.g., as dictated by theregulatory sequences, into proteins within the host cell.

Lentiviral Vector Systems

The invention provides a method of producing a recombinant lentiviruscapable of infecting a dividing or non-dividing cell comprisingtransfecting a suitable host cell with the following: a transfer vectorproviding a nucleic acid encoding a lentiviral gag and a lentiviral pol,where the gag and pol nucleic acid sequences are operably linked to aheterologous regulatory nucleic acid sequence and where the transfervector is defective for nucleic acid sequence encoding functional envprotein and devoid of lentiviral sequences both upstream and downstreamfrom a splice donor site to a gag initiation site of a lentiviralgenome; an envelope construct providing a nucleic acid encoding anon-lentiviral env protein; and a helper construct providing a nucleicacid sequence containing a lentiviral packaging signal flanked bylentiviral cis-acting nucleic acid sequences for reverse transcriptionand integration, and providing a cloning site for introduction of aheterologous nucleic acid sequence operably linked to a regulatorynucleic acid sequence and optionally to a nucleic acid sequence encodingan intercellular trafficking signal, where the nucleic acid sequenceencoding the intercellular trafficking signal is fused in-frame with theheterologous nucleic acid sequence, where the helper construct does notcontain either a complete gag, pol, or env gene, and recovering therecombinant lentivirus. An illustration of the individual vectors usedin the method of the invention is shown in FIG. 1 .

The method of the invention includes the combination of a minimum ofthree vectors in order to produce a recombinant virion or recombinantlentivirus. For example, a vector of the invention can include (a) thep53 gene product, expressed and driven by a regulatory nucleic acidsequence to treat tumor cells or migrating cells having a p53 genemutation; (b) a specific siRNA driven by second regulatory nucleic acidsequence to down-regulate tumor activity of the tumor cells in targettissue or organs; wherein the double or multiple gene system is able toenhance delivery efficacy and therapeutic response. It is understoodthat in the vectors and methods of the present invention, the relativepositions in the transfer vector of the therapeutic molecules—be theyproteins, RNAi or other types of antisense agents—can vary as needed.Therefore, for example, any of the first, second, or third heterologousnucleic acid sequences can encode an RNAi. Furthermore, multipleheterologous nucleic acid sequences (e.g., two or three) can encode anRNAi or other antisense agent.

A first vector is a helper construct, which provides a nucleic acidencoding a lentiviral gag and a lentiviral pol (FIG. 1A).

A second vector is an envelope construct, which provides a nucleic acidencoding a non-lentiviral env protein (FIG. 1B). The env gene can bederived from any virus excluding lentiviruses. The env gene is ideallyderived from a virus other than HIV. The env gene may be amphotropicenvelope protein which allows transduction of cells of human and otherspecies, or may be ecotropic envelope protein, which is able totransduce only mouse and rat cells. Further, it may be desirable totarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Lentiviral vectors can bemade target specific by inserting, for example, a protein. Targeting isoften accomplished by using an antibody to target the lentiviral vector.Those of skill in the art will know of, or can readily ascertain withoutundue experimentation, specific methods to achieve delivery of alentiviral vector to a specific target.

Examples of retroviral-derived env genes include, but are not limitedto: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), and Rous Sarcoma Virus (RSV). Other env genes such as Vesicularstomatitis virus (VSV) (Protein G) can also be used.

The construct providing the viral env nucleic acid sequence is operablyassociated with regulatory sequence, e.g., a promoter or enhancer.Preferably, the regulatory sequence is a viral promoter. The regulatorysequence can be any eukaryotic promoter or enhancer, including forexample, the Moloney murine leukemia virus promoter-enhancer element,the human cytomegalovirus enhancer, or the vaccinia P7.5 promoter. Insome cases, such as the HIV-1 promoter-enhancer element, thesepromoter-enhancer elements are located within or adjacent to the LTRsequences.

A third vector, the transfer vector, provides a nucleic acid sequence,which contains the cis-acting viral sequences necessary for thelentiviral life cycle. Such sequences include the lentiviral psipackaging sequence, reverse transcription signals, integration signals,viral promoter, enhancer, and polyadenylation sequences. The transfervector also contains a cloning site for a heterologous nucleic acidsequence to be transferred to a dividing or non-dividing cell, andoptionally a nucleic acid sequence encoding an intercellular traffickingsignal, where the nucleic acid sequence encoding the intercellulartrafficking signal is fused in-frame with the heterologous nucleic acidsequence (FIG. 1C).

Since recombinant lentiviruses produced by standard methods in the artare defective, they require assistance in order to produce infectiousvector particles. Typically, this assistance is provided, for example,by using a helper cell line that provides the missing viral functions.These plasmids are missing a nucleotide sequence which enables thepackaging mechanism to recognize an RNA transcript for encapsidation.Suitable cell lines produce empty virions, since no genome is packaged.If a lentiviral vector is introduced into such cells in which thepackaging signal is intact, but the structural genes are replaced byother genes of interest, the vector can be packaged and vector virionproduced.

The method of producing the recombinant lentivirus of the invention isdifferent than the standard helper virus/packaging cell line methoddescribed above. The three or more individual vectors used toco-transfect a suitable packaging cell line collectively contain all ofthe required genes for production of a recombinant virus for infectionand transfer of nucleic acid to a non-dividing cell. Consequently, thereis no need for a helper virus.

Conveniently during the cloning stage, the nucleic acid constructreferred to as the transfer vector, having the packaging signal and theheterologous cloning site, also contains a selectable marker gene.Marker genes are utilized to assay for the presence of the vector, andthus, to confirm infection and integration. Typical selection genesencode proteins that confer resistance to antibiotics and other toxicsubstances, e.g. histidinol, puromycin, hygromycin, neomycin,methotrexate, etc.

“Non-dividing” cell refers to a cell that does not go through mitosis.Non-dividing cells may be blocked at any point in the cell cycle, (e.g.,G₀/G₁, G₁/S, G₂/M), as long as the cell is not actively dividing. For exvivo infection, a dividing cell can be treated to block cell division bystandard techniques used by those of skill in the art, including,irradiation, aphidocolin treatment, serum starvation, and contactinhibition. However, it should be understood that ex vivo infection isoften performed without blocking the cells since many cells are alreadyarrested (e.g., stem cells). The recombinant lentivirus vector of theinvention is capable of infecting any non-dividing cell, regardless ofthe mechanism used to block cell division or the point in the cell cycleat which the cell is blocked. Examples of pre-existing non-dividingcells in the body include neuronal, muscle, liver, skin, heart, lung,and bone marrow cells, and their derivatives.

The method of the invention provides at least three vectors whichprovide all of the functions required for packaging of recombinantvirions as discussed above. The method also envisions transfection ofvectors including viral genes such as vpr, vif, nef, vpx, tat, rev, andvpu. Some or all of these genes can be included, for example, on thepackaging construct vector, or, alternatively, they may reside onindividual vectors. There is no limitation to the number of vectorswhich are utilized, as long as they are co-transfected to the packagingcell line in order to produce a single recombinant lentivirus. Forexample, one could put the env nucleic acid sequence on the sameconstruct as the gag and pol.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After co-transfection ofthe at least three vectors to the packaging cell line, the recombinantvirus is recovered from the culture media and titered by standardmethods used by those of skill in the art.

In another embodiment, the invention provides a recombinant lentivirusproduced by the method of the invention as described above.

The invention also provides a method of nucleic acid transfer to anon-dividing cell to provide expression of a particular nucleic acidsequence. Therefore, in another embodiment, the invention provides amethod for introduction and expression of a heterologous nucleic acidsequence in a non-dividing cell comprising infecting the non-dividingcell with the recombinant virus of the invention and expressing theheterologous nucleic acid sequence in the non-dividing cell.

It may be desirable to modulate the expression of a gene regulatingmolecule in a cell by the introduction of a molecule by the method ofthe invention. The term “modulate” envisions the suppression ofexpression of a gene when it is over-expressed, or augmentation ofexpression when it is under-expressed. Where a cell proliferativedisorder is associated with the expression of a gene, nucleic acidsequences that interfere with the gene's expression at the translationallevel can be used. This approach utilizes, for example, antisensenucleic acid, ribozymes, or triplex agents, siRNA to block transcriptionor translation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or triplex agent, or by cleaving it with aribozyme.

The method of the invention may also be useful for neuronal or glialcell transplantation, or “grafting,” which involves transplantation ofcells infected with the recombinant lentivirus of the invention ex vivo,or infection in vivo into the central nervous system or into theventricular cavities or subdurally onto the surface of a host brain.Such methods for grafting will be known to those skilled in the art andare described in Neural Grafting in the Mammalian CNS, Bjorklund andStenevi, eds. (1985). Procedures include intraparenchymaltransplantation, (i.e., within the host brain) achieved by injection ordeposition of tissue within the host brain so as to be apposed to thebrain parenchyma at the time of transplantation.

Self-Inactivating Lentiviral Vectors

Self-inactivating (SIN) lentiviral vectors have a deletion in the U3region of the 3′ LTR that eliminates regulatory sequences, including theTATA box. The deletion has been reported to result in transcriptionalinactivation of the LTR in proviruses without affecting vector titers ortransgene expression in vitro. SIN vectors are described, e.g., byZufferey, et al., 1998, J. Virology 72(12):9873-9880, who made a 400 bpdeletion, and Miyoshi, et al., 1998, J. Virology 72(10):81508157, whomade a 133 bp deletion.

It has been reported that a certain U3 deletion actually results inincreased expression from the vector in vivo (Bayer, et al., 2008, ALarge U3 Deletion Causes Increased In Vivo Expression from aNonintegrating Lentiviral Vector, Molecular Therapydoi:10.1038/mt.2008.199). This finding suggests that additionalalterations to the lentivirus sequences are needed to ensure safety ofgene transfer systems.

II. Heterologous Nucleic Acid Sequences

A heterologous nucleic acid sequence is operably linked to a regulatorynucleic acid sequence. As used herein, the term “heterologous” nucleicacid sequence refers to a sequence that originates from a foreignspecies, or, if from the same species, it may be substantially modifiedfrom its original form. Alternatively, an unchanged nucleic acidsequence that is not normally expressed in a cell is a heterologousnucleic acid sequence. The term “operably linked” refers to functionallinkage between the regulatory sequence and the heterologous nucleicacid sequence. The heterologous sequence can be linked to a promoter.The heterologous nucleic acid sequence can be under control of eitherthe viral LTR promoter-enhancer signals or of an internal promoter, andretained signals within the lentiviral LTR can still bring aboutefficient integration of the vector into the host cell genome. The useof nonintegrating vectors for certain purposes, e.g., where transientexpression is sufficient, is also contemplated.

The recombinant virus of the invention is capable of transferringnucleic acid sequences into a non-dividing cell. The term “nucleic acidsequence” refers to any nucleic acid molecule, preferably DNA. Thenucleic acid molecule may be derived from a variety of sources,including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Suchnucleic acid sequences may comprise genomic DNA which may or may notinclude naturally occurring introns. Moreover, such genomic DNA may beobtained in association with promoter regions, introns, or poly(A)sequences. Genomic DNA may be extracted and purified from suitable cellsby means well known in the art. Alternatively, messenger RNA (mRNA) canbe isolated from cells and used to produce cDNA by reverse transcriptionor other means.

FIG. 2 shows examples of heterologous proteins that can be expressedfrom their genes using the vectors and methods of the present invention.FIGS. 7 and 8 further list examples of cloned structural genes that canserve as, e.g., a first, second, or third heterologous nucleic acidsequence of the invention.

A preferred protein for expression using the vectors and methods of thepresent invention is tumor antigen P53. Expression of P53 is defectivein most cancers, e.g., due to mutation of the gene or loweredexpression. Delivery of the wild-type gene encoding the 53-kilodaltonprotein is therefore a goal of gene therapy for many cancers.

Nucleic acids encoding the same proteins or targeting the same RNAs canbe used in a single transfer vector, for example, two genes for the sameprotein can be cloned from different sources and used as the first andsecond heterologous nucleic acid sequences. Similarly, RNAi sequencesthat are specific for different parts of the same target RNA, or thatdiffer in their percent homology to the target RNA, can be usedtogether.

It may be desirable to transfer a nucleic acid encoding a biologicalresponse modifier. Included in this category are immunopotentiatingagents including nucleic acids encoding a number of the cytokinesclassified as “interleukins.” These include, for example, interleukins 1through 12. Also included in this category, although not necessarilyworking according to the same mechanisms, are interferons, and inparticular gamma interferon (γ-IFN), tumor necrosis factor (TNF) andgranulocyte-macrophage-colony stimulating factor (GM-CSF). It may bedesirable to deliver such nucleic acids to bone marrow cells ormacrophages to treat enzymatic deficiencies or immune defects, or cancerdisease. Nucleic acids encoding growth factors, toxic peptides, ligands,receptors, or other physiologically important proteins can also beintroduced into specific non-dividing cells.

Selection of RNAi Agents and Other Antisense Nucleic Acid Sequences

An RNAi agent used in the vectors and methods of the present inventioncan be targeted to any RNA molecule. Besides messenger RNA (mRNA), RNAiagents can target, e.g., various species of microRNA. The use of RNAi ingene therapy and RNAi selection and sequence design, are described,e.g., in WO 2007/109131, “Lentiviral Vectors That Provide ImprovedExpression and Reduced Variegation after Transgenesis,” and WO2007/087113, “Natural Antisense and Non-Coding RNA Transcripts as DrugTargets,” both of which are incorporated herein by reference.

It may be desirable to modulate the expression of a gene regulatingmolecule in a cell by the introduction of a molecule by the method ofthe invention. The term “modulate” envisions the suppression ofexpression of a gene when it is over-expressed, or augmentation ofexpression when it is under-expressed. Where a cell proliferativedisorder is associated with the expression of a gene, nucleic acidsequences that interfere with the gene's expression at the translationallevel can be used. This approach utilizes, for example, antisensenucleic acid, ribozymes, or triplex agents, siRNA to block transcriptionor translation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or triplex agent, or by cleaving it with aribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, 1990Scientific American 262:40). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate an mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target cell. The use of antisensemethods to inhibit the in vitro translation of genes is well known inthe art (Marcus-Sakura, 1988 Anal Biochem 172:289).

The antisense nucleic acid can be used to block expression of a mutantprotein or a dominantly active gene product, such as amyloid precursorprotein that accumulates in Alzheimer's disease. Such methods are alsouseful for the treatment of Huntington's disease, hereditaryParkinsonism, and other diseases. Antisense nucleic acids are alsouseful for the inhibition of expression of proteins associated withtoxicity.

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al. 1991Antisense Res and Dev 1:227; Helene, C. 1991 Anticancer Drug Design6:569).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, 1988 J Amer Med Assn 260:3030). A major advantage ofthis approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

RNA interference (RNAi) is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their target nucleicacid sequences (Caplen, N. J., et al, Proc. Natl. Acad. ScL USA98:9742-9747 (2001)). Biochemical studies in Drosophila cell-freelysates indicate that, in certain embodiments of the present invention,the mediators of RNA-dependent gene silencing are 21-25 nucleotide“small interfering” RNA duplexes (siRNAs). The siRNAs are derived fromthe processing of dsRNA by an RNase enzyme known as Dicer (Bernstein,E., et al, Nature 409:363-366 (2001)). siRNA duplex products arerecruited into a multi-protein siRNA complex termed RISC (RNA InducedSilencing Complex). Without wishing to be bound by any particulartheory, a RISC is then believed to be guided to a target nucleic acid(suitably mRNA), where the siRNA duplex interacts in a sequence-specificway to mediate cleavage in a catalytic fashion (Bernstein, E., et al,Nature 409:363-366 (2001); Boutla, A., et al, Curr. Biol. 11:1776-1780(2001)). Small interfering RNAs that can be used in accordance with thepresent invention can be synthesized and used according to proceduresthat are well known in the art and that will be familiar to theordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 0 toabout 50 nucleotides (nt). In examples of nonlimiting embodiments,siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

“RNAi” or “RNAi agent” refers to an at least partly double-stranded RNAhaving a structure characteristic of molecules that are known in the artto mediate inhibition of gene expression through an RNAi mechanism or anRNA strand comprising at least partially complementary portions thathybridize to one another to form such a structure. When an RNA comprisescomplementary regions that hybridize with each other, the RNA will besaid to self-hybridize. An RNAi agent includes a portion that issubstantially complementary to a target gene. An RNAi agent, optionallyincludes one or more nucleotide analogs or modifications. One ofordinary skill in the art will recognize that RNAi agents that aresynthesized in vitro can include ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides or backbones, etc., whereasRNAi agents synthesized intracellularly, e.g., encoded by DNA templates,typically consist of RNA, which may be modified following transcription.Of particular interest herein are short RNAi agents, i.e., RNAi agentsconsisting of one or more strands that hybridize or self-hybridize toform a structure that comprises a duplex portion between about 15-29nucleotides in length, optionally having one or more mismatched orunpaired nucleotides within the duplex. RNAi agents include shortinterfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and other RNAspecies that can be processed intracellularly to produce shRNAsincluding, but not limited to, RNA species identical to a naturallyoccurring miRNA precursor or a designed precursor of an miRNA-like RNA.

The term “short, interfering RNA” (siRNA) refers to a nucleic acid thatincludes a double-stranded portion between about 15-29 nucleotides inlength and optionally further comprises a single-stranded overhang(e.g., 1-6 nucleotides in length) on either or both strands. Thedouble-stranded portion is typically between 17-21 nucleotides inlength, e.g., 19 nucleotides in length. The overhangs are typicallypresent on the 3′ end of each strand, are usually 2 nucleotides long,and are composed of DNA or nucleotide analogs. An siRNA may be formedfrom two RNA strands that hybridize together, or may alternatively begenerated from a longer double-stranded RNA or from a single RNA strandthat includes a self-hybridizing portion, such as a short hairpin RNA.One of ordinary skill in the art will appreciate that one or more,mismatches or unpaired nucleotides can be present in the duplex formedby the two siRNA strands. One strand of an siRNA (the “antisense” or“guide” strand) includes a portion that hybridizes with a target nucleicacid, e.g., an mRNA transcript. Typically the antisense strand isperfectly complementary to the target over about 15-29 nucleotides,typically between 17-21 nucleotides, e.g., 19 nucleotides, meaning thatthe siRNA hybridizes to the target transcript without a single mismatchover this length. However, one of ordinary skill in the art willappreciate that one or more mismatches or unpaired nucleotides may bepresent in a duplex formed between the siRNA strand and the targettranscript.

“Short hairpin RNA” refers to a nucleic acid molecule comprising atleast two complementary portions hybridized or capable of hybridizing toform a duplex structure sufficiently long to mediate RNAi (typicallybetween 15-29 nucleotides in length), and at least one single-strandedportion, typically between approximately 1 and 10 nucleotides in lengththat forms a loop connecting the ends of the two sequences that form theduplex. The structure may further comprise an overhang. The duplexformed by hybridization of self-complementary portions of the shRNA hassimilar properties to those of siRNAs and, as described below, shRNAsare processed into siRNAs by the conserved cellular RNAi machinery. ThusshRNAs are precursors of siRNAs and are similarly capable of inhibitingexpression of a target transcript. As is the case for siRNA, an shRNAincludes a portion that hybridizes with a target nucleic acid, e.g., anmRNA transcript and is usually the perfectly complementary to the targetover about 15-29 nucleotides, typically between 17-21 nucleotides, e.g.,19 nucleotides. However, one of ordinary skill in the art willappreciate that one or more mismatches or unpaired nucleotides may bepresent in a duplex formed between the shRNA strand and the targettranscript.

An RNAi agent is considered to be “targeted” to a transcript and to thegene that encodes the transcript if (1) the RNAi agent comprises aportion, e.g., a strand, that is at least approximately 80%,approximately 85%, approximately 90%, approximately 91%, approximately92%, approximately 93%, approximately 94%, approximately 95%,approximately 96%, approximately 97%, approximately 98%, approximately99%, or approximately 100% complementary to the transcript over a regionabout 15-29 nucleotides in length, e.g., a region at least approximately15, approximately 17, approximately 18, or approximately 19 nucleotidesin length; and/or (2) the Tm of a duplex formed by a stretch of 15nucleotides of one strand of the RNAi agent and a 15 nucleotide portionof the transcript, under conditions (excluding temperature) typicallyfound within the cytoplasm or nucleus of mammalian cells and/or in aDrosophila lysate as described, e.g., in U.S. Patent App. Pubs.2002/0086356 and 2004/0229266, is no more than approximately 15° C.lower or no more than approximately 10° C. lower, than the Tm of aduplex that would be formed by the same 15 nucleotides of the RNAi agentand its exact complement; and/or (3) the stability of the transcript isreduced in the presence of the RNAi agent as compared with its absence.An RNAi agent targeted to a transcript is also considered targeted tothe gene that encodes and directs synthesis of the transcript. A “targetregion” is a region of a target transcript that hybridizes with anantisense strand of an RNAi agent. A “target transcript” is any RNA thatis a target for inhibition by RNA interference. The terms “target RNA”and “target transcript” are used interchangeably herein.

Selection of appropriate RNAi agents is facilitated by using computerprograms that automatically align nucleic acid sequences and indicateregions of identity or homology. Such programs are used to comparenucleic acid sequences obtained, for example, by searching databasessuch as GenBank or by sequencing PCR products. Comparison of nucleicacid sequences from a range of species allows the selection of nucleicacid sequences that display an appropriate degree of identity betweenspecies. In the case of genes that have not been sequenced, Southernblots are performed to allow a determination of the degree of identitybetween genes in target species and other species. By performingSouthern blots at varying degrees of stringency, as is well known in theart, it is possible to obtain an approximate measure of identity. Theseprocedures allow the selection of RNAi that exhibit a high degree ofcomplementarity to target nucleic acid sequences in a subject to becontrolled and a lower degree of complementarity to correspondingnucleic acid sequences in other species. One skilled in the art willrealize that there is considerable latitude in selecting appropriateregions of genes for use in the present invention.

Selection of an appropriate antisense nucleic acid is facilitated byusing computer programs that automatically align nucleic acid sequencesand indicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots can be performed to allow a determination of the degreeof identity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of antisense nucleicacids that exhibit a high degree of complementarity to target nucleicacid sequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

Selection of hybridization sites for antisense nucleic acids can be madeby one of skill in the art using methods described in the literature.For example, Ding, et al., report a method for defining mRNAhybridization sites based on determining RNA structures using algorithmsand thermodynamic and structural properties of the RNA (Ding, et al.,2001, Statistical prediction of single-stranded regions in RNA secondarystructure and application to predicting effective antisense target sitesand beyond, Nucleic Acids Research 29(5):1034-1046; incorporated hereinby reference in its entirety). Sczakiel, et al., also describe a methodfor computer-supported design of antisense oligonucleotides (Sczakiel,et al., 2000, Theoretical and experimental approaches to designeffective antisense oligonucleotides, Frontiers in Bioscience 5:D194-201; Scherr, et al., 2000, RNA accessibility prediction: atheoretical approach is consistent with experimental studies in cellextracts, Nucleic Acids Research 28: 2455-2461; Patzel, et al., 1999, Atheoretical approach to select effective antisenseoligodeoxyribonucleotides at high statistical probability, J. Biol.Chem. 266:18162-18171; all incorporated herein by reference in theirentirety).

Reports of other methods for identifying mRNA hybridization sites usedinclude, e.g., Chiang, et al., who describe a method based oncalculating melting temperatures (Chiang, et al., 1991, Antisenseoligonucleotides inhibit intercellular adhesion molecule 1 expression bytwo distinct mechanisms, J. Biol. Chem. 266: 18162-18171, incorporatedherein by reference in its entirety). Methods based on calculation ofduplex formation free energies have been used (see, e.g., Stull, et al.,1992, Predicting antisense oligonucleotide inhibitory efficacy: acomputational approach using histograms and thermodynamic indices,Nucleic Acids Research 20:3501-3508; Ding, et al., 1999, A bayesianstatistical algorithm for RNA secondary structure prediction, Comput.Chem. 23:387-400; all incorporated herein by reference in theirentirety). Still other methods rely on the use of combinatorialoligonucleotides to identify the hybridization sites within the targetRNA. Identification of the hybridization sites is made using RNase Hcleavage (Lloyd, et al., 2001, Determination of optimal sites ofantisense oligonucleotide cleavage within TNFα mRNA, Nucleic AcidsResearch 29:3664-3673, incorporated herein by reference in itsentirety), microarray analysis (Mir, et al., 1999, Determining theinfluence of structure on hybridization using oligonucleotide arrays,Nature Biotechnology 17:788-792; and Sohail, et al., 2001, Antisenseoligonucleotides selected by hybridization to scanning arrays areeffective reagents in vivo, Nucleic Acids Research 29:2041-2051, bothincorporated herein by reference in their entirety) or MALDI-TOF massspectrometry (Altman, et al., 1999, Selection of modifiedoligonucleotides with increased target affinity via MALDI-monitorednuclease survival assays, J. Comb. Chem. 1:493-508, incorporated hereinby reference in its entirety).

The utility of an antisense nucleic acid molecule for modulation(including inhibition) of an mRNA can be readily determined by simpletesting. Thus, an in vitro or in vivo expression system comprising thetargeted mRNA, mutations or fragments thereof, can be contacted with aparticular antisense nucleic acid molecule (modified or unmodified) andlevels of expression are compared to a control, that is, using theidentical expression system which was not contacted with the antisensenucleic acid molecule. In vitro assays of oligonucleotide activity canalso be useful for identifying antisense nucleic acids of the invention.For example, Lloyd, et al., report a direct inverse correlation betweenpredicted chimeric antisense oligonucleotide activities, as determinedusing an in vitro RNase H assay, and the resultant levels of mRNA andprotein expression (Lloyd, et al., 2001, Determination of optimal sitesof antisense oligonucleotide cleavage within TNFα mRNA, Nucleic AcidsResearch 29(17): 3664-3673). According to Lloyd, et al., the ability ofthe in vitro assay to predict oligonucleotide efficacy was superior toother computationally based RNA structural predictions, ΔG calculationsand in vivo trial and error methodologies.

Bc1-2 and molecules that work in conjunction with Bc1-2 are also targetsfor cancer therapy. B cell leukemia/lymphoma-2 (Bc1-2) is the prototypemember of a family of cell death regulatory proteins. Bc1-2 is foundmainly in the mitochondria and blocks apoptosis by interfering with theactivation of caspases. Gene transfer of Bc1-2 into tumor cells has beenshown to enhance their metastatic potential (Miyake et al., 1999). Bc1-2gene transfer may be applied to bone marrow transplant since Bc1-2enhances the survival of hematopoietic stem cells after reconstitutionof irradiated recipient (Innes et al., 1999). Also, Bc1-2 gene transfercould be useful against neurodegenerating diseases since expression ofBc1-2 in neurons protects them from apoptosis (Saille et al., 1999).Bcl-XS (short isoform) is a dominant negative repressor of Bc1-2 andBcl-XL. It has been used in gene therapy experiments to initiateapoptosis in tumors that express Bc1-2 and Bcl-XL. Expression of Bcl-XSreduces tumor size (Ealovega et al., 1996) and sensitizes tumor cells tochemotherapeutic agents (Sumatran et al., 1995), suggesting a role forBcl-XS in initiating cell death in tumors that express Bc1-2 or Bcl-XL(Dole et al., 1996). Expression of these genes or RNAi agents targetingthem can be selected as appropriate for the condition being treated.

Equivalent Molecules

The invention comprehends that the therapeutic molecules delivered usingthe vectors and methods of the present invention can be modified. Thenucleic acid molecule encoding a given protein be modified, forinstance, due to the degeneracy of codon usage, a coding sequence can bemodified, and modified and truncated forms of a protein can be used,such as those which may be found in the literature or analogous totruncated or modified forms found in the literature.

Likewise, analogs, homologs, derivatives, and variants of the codingsequences can be used and analogs, homologs, derivatives and variants ofproteins can be expressed; such expressed analogs, homologs, derivativesand variants of proteins can have activity analogous to that of thefull-length protein, and the analogs, homologs, derivatives and variantsof the protein coding sequence encode such active analogs, homologs,derivatives, and variants.

III. Heterologous Regulatory Sequences

A “first heterologous regulatory sequence” is positioned upstream (5′of) the first heterologous nucleic acid sequence, encoding, e.g., atherapeutic gene. Similarly, a “second heterologous regulatory sequence”can be positioned upstream (5′ of) the second heterologous nucleic acidsequence and downstream of the first heterologous nucleic acid sequence,and a “third heterologous regulatory sequence” can be positionedupstream (5′ of) the third heterologous nucleic acid sequence anddownstream of the second heterologous nucleic acid sequence. Aheterologous regulatory sequence can be a sequence that influences,e.g., expression or localization, of a therapeutic molecule encoded by aheterologous nucleic acid sequence. A heterologous regulatory sequencecan comprise a promoter, enhancer, protease recognition (cleavage)sequence, internal ribosome binding site, intracellular or intercellulartrafficking (transport) signal, etc. Certain heterologous regulatorysequences, e.g., cleavage sequences and trafficking sequences, can beexpressed as part of a fusion with a therapeutic molecule.

In embodiments, a heterologous regulatory sequence affects an upstreamheterologous nucleic acid sequence. Therefore, a fourth regulatorysequence, located downstream of a third heterologous nucleic acidsequence, can be included in the transfer vector. Also, for example, thesecond heterologous regulatory sequence can contain elements that affectexpression or localization of the first heterologous nucleic acid andits corresponding therapeutic molecule, and the third heterologousregulatory sequence can contain elements that affect expression orlocalization of the second heterologous nucleic acid and itscorresponding therapeutic molecule.

Promoters and Enhancers

The promoter sequence may be homologous or heterologous to the desiredgene sequence. A wide range of promoters may be utilized, includingviral or mammalian promoters. Cell or tissue specific promoters can beutilized to target expression of gene sequences in specific cellpopulations. Suitable mammalian and viral promoters for the presentinvention are available in the art.

Examples of promoters, cellular promoters/enhancers and induciblepromoters/enhancers that can be used in combination with the presentinvention are listed in FIGS. 4 and 5 . Any suitable promoter/enhancercombination (as per the Eukaryotic Promoter Database, EPDB) can be usedto drive expression of constructs of the invention.

Trafficking Signals

Trafficking signals can direct a molecule to different compartmentswithin a cell, as well as outside the cell and into other cells. An“intercellular trafficking signal,” or transport signal, is an aminoacid sequence that imparts the property to a protein of being able topass through membranes between cells. Examples of membrane-penetratingproteins include, but are not limited to, several plant and bacterialprotein toxins, such as ricin, abrin, modeccin, diphtheria toxin,cholera toxin, anthrax toxin, heat labile toxins, and Pseudomonasaeruginosa exotoxin A. Examples of membrane-penetrating proteins thatare not toxins include the TAT protein of human immunodeficiency virusand the protein VP22, the product of the UL49 gene of herpes simplexvirus type 1. One line of research involves adapting such molecules fromtheir naturally destructive role into therapeutic compositions.

The effectiveness of lentiviral vectors to deliver genes encodingproteins fused to herpes simplex virus type 1 tegument protein VP22 hasbeen reported (see, e.g., U.S. Pat. App. Pub. No. 2003/0119770). “VP22”denotes: protein VP22 of HSV, e.g., of HSV1, and transport-activefragments and homologues thereof, including transport-active homologuesfrom other herpesviruses including varicella zoster virus VZV, marek'sdisease virus MDV and bovine herpesvirus BHV.

Among sub-sequences of herpesviral VP22 protein with transport activity,investigators have found that, for example, transport activity ispresent in polypeptides corresponding to amino acids 60-301 and 159-301of the full HSV1 VP22 sequence (1-301). A polypeptide consisting of aa175-301 of the VP22 sequence has markedly less transport activity, andis less preferred in connection with the present invention. Accordingly,the present invention relates in one aspect to a sub-sequence of VP22containing a sequence starting preferably from about aa 159 (or earlier,towards the N-terminal, in the native VP22 sequence), to about aa 301,and having (relative to the full VP22 sequence) at least one deletion ofat least part of the VP22 sequence which can extend for example from theN-terminal to the cited starting point, e.g., a deletion of all or partof the sequence of about aa 1-158. (Less preferably, such a deletion canextend further in the C-terminal direction, e.g., to about aa 175.) Forexample, partial sequences in the range from about aa 60-301 to about aa159-301 are provided.

VP22 sequences as contemplated herein extend to homologous proteins andfragments based on sequences of VP22 protein homologues from otherherpesviruses, e.g., the invention provides corresponding derivativesand uses of the known VP22-homologue sequences from VZV (e.g., all orhomologous parts of the sequence from aa 1-302), from MDV (e.g., all orhomologous parts of the sequence from aa 1-249) and from BHV (e.g., allor homologous parts of the sequence from aa 1-258). The sequences of thecorresponding proteins from HSV2, VZV, BHV and MDV are available inpublic protein/nucleic acid sequence databases. Thus, for example,within the EMBL/Genbank database, a VP22 sequence from HSV2 is availableas gene item UL49 under accession no. Z86099 containing the completegenome of HSV2 strain HG52; the complete genome of VZV including thehomologous gene/protein is available under accession numbers X04370,M14891, M16612; the corresponding protein sequence from BHV is availableas “bovine herpesvirus 1 virion tegument protein” under accession numberU21137; and the corresponding sequence from MDV is available as geneitem UL49 under accession number L10283 for “gallid herpesvirus type 1homologous sequence genes”. In these proteins, especially those fromHSV2 and VZV, corresponding deletions can be made, e.g., of sequenceshomologous to aa 1-159 of VP22 from HSV1. Homologies between thesesequences are readily accessible by the use of standard algorithms,default parameters, and software.

Furthermore, chimeric VP22 proteins and protein sequences are alsouseful within the context of the present invention, e.g., a proteinsequence from VP22 of HSV1 for part of which a homologous sequence fromthe corresponding VP22 homologue of another herpesvirus has beensubstituted. For example, into the sequence of polypeptide 159-301 fromVP22 of HSV1, C-terminal sequences can be substituted from VP22 of HSV2or from the VP22 homologue of BHV.

Investigators have found that deletion of the 34-amino acid C-terminalsequence from VP22 of HSV1 abolishes transport-activity, thus thissequence region contains essential elements for transport activity.According to a further aspect of the invention, there are providedin-frame fusions comprising a nucleic acid sequence encoding the34-amino acid C-terminal sequence from VP22, or a variant thereof,together with a sequence for a heterologous nucleic acid sequence.In-frame fusions of nucleic acid sequences encoding modified terminalfragments having at least one mutation insertion or deletion relative tothe C-terminal 34 amino acid sequence of HSV1 VP22 are also provided.

Investigators have also been found that sequences necessary fortransport activity contain one or a plurality of amino acid sequencemotifs or their homologues from the C-terminal sequence of VP22 of HSV1or other herpesviruses, which can be selected from RSASR (SEQ ID NO: 1),RTASR (SEQ ID NO: 2), RSRAR (SEQ ID NO: 3), RTRAR (SEQ ID NO: 4), ATATR(SEQ ID NO 5), and wherein the third or fourth residue A can beduplicated, e.g., as in RSAASR (SEQ ID NO: 6). Corresponding in-framefusions of nucleic acid sequences encoding these signals are alsoprovided.

The HIV-1 Tat protein was also reported to enhance intercellulartrafficking in vitro. It is composed of 86 amino acids and contains ahighly basic region and a cysteine-rich region. It was found thatTat-derived peptides as short as eleven amino acids are sufficient fortransduction of proteins (Fawell, et al., 1994, Proc Natl Acad Sci USA91:664-668). However, the exact mechanism by which the 11-amino acidtransduction domain crosses lipid bilayers is poorly understood.Schwarze et al. (Science, 385:1569-1572, 1999) reported generating aTat-β-galactosidase fusion protein that was delivered efficiently intobrain tissue and skeletal muscle in vivo.

In embodiments of the present invention, a Tat-derived traffickingprotein of eleven amino acids Try-Gly-Arg-Lys-lys-Arg-Arg-Gln-Arg-Arg(SEQ ID NO: 7) is used to enhance intercellular trafficking oftherapeutic molecules. Any appropriate nucleic acid sequence can be usedto express this protein, e.g., tat ggc agg aag aag cgg aga cag cga cgaaga (SEQ ID NO:8) with a start codon.

Control of intracellular as well as intercellular transport iscontemplated for use in the methods of the invention. This level ofcontrol can be used to target therapeutic proteins to particularcellular compartments, e.g., to correct defects for proteins involved inspecific disease processes.

Other potentially useful Tat-derived trafficking sequences have beendescribed. For example Chauhan, et al., 2007, The Taming of the CellPenetrating Domain of the HIV Tat: Myths and Realities, J. ControlRelease 117(2): 148-162, incorporated herein by reference in itsentirety, disclose variants of the Tat protein transduction domain.

Following are Tat-derived cell penetrating peptides described byChauhan, et al.:

(SEQ ID NO: 9) PTD YGRKKRRQRRR (SEQ ID NO: 10) PTD-4 YARAAARQARA(SEQ ID NO: 11) YM-3 THRLPRRRRRR (SEQ ID NO: 12) CTP GGRRARRRRRR

As reported by the authors, depending on the nature of the protein beingtransported, these peptides effect transport, including transport amongcellular compartments. For example, cytoplasmic proteins reportedly endup in the nucleus when PTD is used, nucleo-cytoplasmic proteins go tothe nucleus when PTD-4 is used, secretory proteins are found in thenucleus and outside the cell when YM-3 is used, and membrane proteins goto the membrane and nucleus when CTP is used.

Other cell penetrating proteins useful for introducing recombinantproteins into cells are penetratin, polylysine, polyarginine, KaposiFGF, Syn B1, FGF-4, nuclear localization signal, anthrax toxinderivative 254-amino acids peptide segment, diphtheria toxin “R” bindingdomain, MPG (described below), WR peptide, and exotoxin A. Penetratinpeptide has also been used for siRNA delivery to cells. In embodiments,these proteins or their derivatives used in as trafficking signals inthe vectors and methods of the invention.

A fusion peptide, “MPG,” has been described for efficient transductionof nucleic acids. This peptide is a bipartite amphipathic peptideobtained by combining the fusion domain of HIV-gp41 protein and the NLSdomain of SV40 large T antigen. This peptide is being used as ananoparticle for transduction of siRNA in vitro and is also availablecommercially. (See, e.g., Chauhan, et al., 2007; Morris, et al., 1999, Anovel potent strategy for gene delivery using a single peptide vector asa carrier, Nucleic Acids Research 27:3510-3517.)

In embodiments of the vectors and methods of the invention, an siRNA orother antisense agent is transported intercellularly or intracellularly.

The inventive transfer vector may include splice acceptor and splicedonor sequences flanking the gene construct. In the case of multigenetransfer vector, the splice acceptor sequence may be inserted upstreamof the first promoter-structural gene unit and splice donor site may belocated in the downstream of the last promoter-structural gene unit inthe 5′ to 3′ order. A mammalian insulator sequence (MIS) may be inserteddownstream of the gene units, or if specific promoters are used for thegene units, then the MIS may be placed before the first gene unit andafter the last gene unit followed by splice donor site. A translationinitiation sequence may also be included in the multigene gene transfervector. The use of the translation initiation sequence causes the secondand subsequent multigene units to be expressed evenly and stablycompared with the first gene expression product.

Translation Initiation Sequence

The transfer vector may optionally comprise a sequence that allows fortranslation initiation in the middle of a messenger RNA (mRNA) sequenceas part of the greater process of protein synthesis. While this sequencemay be typically IRES other sequences may be used, which may have asimilar sequence.

Splice Acceptor/Splice Donor Sequence

The transfer vector may optionally comprise a promoter-gene sequenceflanked by a splice acceptor site 5′ to a gene unit and a splice donorsite 3′ to the promoter-gene. For instance, the following elements maybe present 5′ to 3′: a splice acceptor site, first promoter for thefirst gene unit, first heterologous nucleic acid sequence and splicedonor site 3′ to the first gene unit, then a splice acceptor site and asecond promoter for the second gene, the second heterologous nucleicacid sequence, splice donor site, and then if there is a third gene, asplice acceptor site, third promoter for the third gene unit, the thirdheterologous nucleic acid sequence, and a splice donor site, and soforth. The splice acceptor or donor site typically may include about 5to 10 bases.

Mammalian Insulator Sequence (MIS)

The transfer vector may optionally comprise an insulator sequence, inparticular mammalian insulator sequence (MIS). Insulators are DNAsequence elements that can protect against the activation influence ofdistal enhancers associated with other genes, and also help to preservethe independent function of genes embedded in a genome in which they aresurrounded by regulatory signals so that cross interaction is avoided.The insulators as used in the present application may not necessarily belimited to use in lentiviruses. The insulators may be used with otherviral vectors.

To provide background on these insulator sequences, the zinc fingerprotein CCCTC-binding factor (CTCF) is a versatile transcriptionregulator that binds to insulators and shows enhancer-blocking activityfor regulating gene expression control. Chicken (beta-globin) insulatorwith about 1.2 kb is widely used in vitro or in vivo animals, butgenerally does not have human or mammalian compatible factors to beentirely useful in treating humans or mammals.

Bovine or human growth hormone transcriptional stop sequences may alsobe used as insulators. A short-element of about 238 bp containing the“HS” DNA element core sequence, which is the binding site for CTCF isalso effective as an “enhancer blocking” element. Template DNA forgeneration of such element, for example a pcDNA3, which contains bovinegrowth hormone transcriptional stop sequence may be as follows: PCRprimers: 5′ agctagatagtgtcacctaaatgc-3′ (SEQ ID NO:13) and5′-agcatgcctgctatt-3′ (SEQ ID NO:14).

A binding site for the transcription factor CTCF may be responsible forenhancer-blocking activity in a variety of insulators, including theinsulators at the 5′ and 3′ chromatin boundaries of the chicken andhuman beta-globin locus. The minimal element responsible for thisactivity may be a binding site for CTCF.

When several different sequences of insulators of 5′ and 3′ HS/CTCF arecompared using “human insulator” as the template, there are at least twomutations in the mouse, at least 5 mutations in chicken at the 5′HS/CTCF. In addition, there are at least two mutations in mouse, atleast 4 mutations at the 3′ HS/CTCF.

The insulator sequence as used in the lentivirus transfer vector may beplaced as follows. In a single gene vector where a general promoter isused to control gene expression, MIS may be placed 3′ of the gene andupstream of the splicing donor site. However, if a specific promoter isused to control the expression of the gene, the MIS may be placedupstream of the specific promoter and downstream of the gene, whereinthe MIS sequences are optionally flanked by the splice acceptor on the5′ side and splice donor on the 3′ side of the gene. In a single geneconstruct, for RNAi expression where specific promoter is used, MIS mayflank the specific promoter-structural gene construct, optionally with asplice donor site downstream of the 3′ MIS. Two or more MIS may be usedtogether to enhance the blocking effects.

For multiple gene vectors, if a general promoter is used, an MIS may beincluded downstream of the gene units with splice donor site 3′ to theMIS. However, if a specific promoter is used MIS is place upstream ofthe first specific promoter for the first structural gene, and anotherMIS downstream of the last specific promoter-structural gene set. TheMIS are optionally flanked by splice acceptor on the 5′ side and splicedonor on the 3′ side.

IV. Therapeutic Applications

The invention includes a variety of therapeutic applications for thelentiviral vectors of the invention. In particular, lentiviral vectorsare useful for gene therapy. Exemplary therapeutic applications arelisted in FIG. 7 . The invention provides methods of treating and/orpreventing infection by an infectious agent, the method comprisingadministering to a subject prior to, simultaneously with, or afterexposure of the subject to the infectious agent a composition comprisingan effective amount of a lentiviral vector, wherein the lentiviralvector directs transcription of at least one RNA that hybridizes to forman shRNA or siRNA that is targeted to a transcript produced duringinfection by the infectious agent, which transcript is characterized inthat reduction in levels of the transcript delays, prevents, and/orinhibits one or more aspects of infection by and/or replication of theinfectious agent.

The invention provides methods of treating a disease or clinicalcondition by, for example, removing a population of cells from a subjectat risk of or suffering from the disease or clinical condition andengineering or manipulating the cells to comprise an effective amount oftherapeutic agents by infecting or transfecting the cells with alentiviral vector. At least a portion of the cells are returned to thesubject.

Without limitation, therapeutic approaches may find particular use indiseases such as cancer, in which a mutation in a cellular gene isresponsible for or contributes to the pathogenesis of the disease, andin which specific inhibition of the target transcript bearing themutation may be achieved by expressing an RNAi agent targeted to thetarget transcript within the cells, without interfering with expressionof the normal (i.e. non-mutated) allele. Furthermore, treatment of anycancer in which P53 expression is defective is contemplated using thevectors and methods of the invention.

The invention is also useful for the treatment of genetic diseases, forexample, Gaucher's Disease and Fabry's Disease. Gaucher's disease is alysosomal storage disease caused by a deficiency of the enzymeglucocerebrosidase. This deficiency leads to an accumulation of theenzyme substrate, the fatty substance glucocerebroside (also known asglucosylceramide). Fatty material can collect in the spleen, liver,kidneys, lungs, brain and bone marrow. It has been reported, using amouse model for Gaucher's Disease, that a lentiviral vector cantransduce HSCs that are capable of long-term gene expression in vivo(Kim, et al., 2005, “Long-term expression of the humanglucocerebrosidase gene in vivo after transplantation ofbone-marrow-derived cells transformed with a lentivirus vector, J. GeneMed. 7:878-887, incorporated herein by reference).

According to certain embodiments of the invention, rather than removingcells from the body of a subject, infecting or transfecting them intissue culture, and then returning them to the subject, inventivelentiviral vectors or lentiviruses are delivered directly to thesubject.

Neurological Disorders

Cells infected with a recombinant lentivirus of the invention, in vivo,or ex vivo, used for treatment of a neuronal disorder for example, mayoptionally contain an exogenous gene, for example, a gene which encodesa receptor or a gene which encodes a ligand. Such receptors includereceptors which respond to dopamine, GABA, adrenaline, noradrenaline,serotonin, glutamate, acetylcholine and other neuropeptides, asdescribed above. Examples of ligands which may provide a therapeuticeffect in a neuronal disorder include dopamine, adrenaline,noradrenaline, acetylcholine, gamma-aminobutyric acid and serotonin. Thediffusion and uptake of a required ligand after secretion by an infecteddonor cell would be beneficial in a disorder where the subject's neuralcell is defective in the production of such a gene product. A cellgenetically modified to secrete a neurotrophic factor, such as nervegrowth factor (NGF), might be used to prevent degeneration ofcholinergic neurons that might otherwise die without treatment.Alternatively, cells can be grafted into a subject with a disorder ofthe basal ganglia, such as Parkinson's disease, or can be modified tocontain an exogenous gene encoding L-DOPA, the precursor to dopamine.Parkinson's disease is characterized by a loss of dopamine neurons inthe substantia nigra of the midbrain, which have the basal ganglia astheir major target organ.

U.S. Pat. No. 6,800,281, “Lentiviral-mediated growth factor gene therapyfor neurodegenerative diseases,” incorporated herein by reference in itsentirety, describes methods for treating Parkinson's Disease using glialcell derived neurotrophic factor (GDNF), highly conserved neurotrophicfactor that potently promotes the survival of many types of neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterizedby the loss of the nigrostriatal pathway; a progressive disorderresulting from degeneration of dopaminergic neurons within thesubstantia nigra. Although the cause of Parkinson's disease is notknown, it is associated with the progressive death of dopaminergic(tyrosine hydroxylase (TH) positive) mesencephalic neurons, inducingmotor impairment. The characteristic symptoms of Parkinson's diseaseappear when up to 70% of TH-positive nigrostriatal neurons havedegenerated. Surgical therapies aimed at replacing lost dopaminergicneurons or disrupting aberrant basal ganglia circuitry have recentlybeen tested (C. Honey et al. 1999). However, these clinical trials havefocused on patients with advanced disease, and the primary goal offorestalling disease progression in newly diagnosed patients has yet tobe realized. The administration can be by stereotaxic injection. Theadministration can be intracranially, e.g., intracranially to striatumor to substantia nigra. The administration can also be by retrogradetransport.

In an embodiment, the administration site is the striatum of the brain,in particular the caudate putamen. Injection into the putamen can labeltarget sites located in various distant regions of the brain, forexample, the globus pallidus, amygdala, subthalamic nucleus or thesubstantia nigra. Transduction of cells in the pallidus commonly causesretrograde labelling of cells in the thalamus. In a preferred embodimentthe (or one of the) target site(s) is the substantia nigra.

In another embodiment the vector system is injected directly into thespinal cord. This administration site accesses distal connections in thebrain stem and cortex. Within a given target site, the vector system maytransduce a target cell. The target cell may be a cell found in nervoustissue, such as a neuron, astrocyte, oligodendrocyte, microglia orependymal cell. In a preferred embodiment, the target cell is a neuron,in particular a TH positive neuron.

The vector system can be administered by direct injection. Methods forinjection into the brain (in particular the striatum) are well known inthe art (Bilang-Bleuel et al (1997) Proc. Acad. Nati. Sci. USA94:8818-8823; Choi-Lundberg et al (1998) Exp. Neuro1.154:261-275;Choi-Lundberg et al (1997) Science 275:838-841; and Mandel et al (1997))Proc. Acad. Natl. Sci. USA 94:14083-14088). Stereotaxic injections maybegiven.

Administration of the cells or virus into selected regions of therecipient subject's brain may be made by drilling a hole and piercingthe dura to permit the needle of a microsyringe to be inserted. Thecells or recombinant lentivirus can alternatively be injectedintrathecally into the spinal cord region. A cell preparation infectedex vivo, or the recombinant lentivirus of the invention, permitsgrafting of neuronal cells to any predetermined site in the brain orspinal cord, and allows multiple grafting simultaneously in severaldifferent sites using the same cell suspension or viral suspension andpermits mixtures of cells from different anatomical regions.

For transduction in tissues such as the brain, it is necessary to usevery small volumes, so the viral preparation is concentrated byultracentrifugation. The resulting preparation should have at least 10⁸pfu./ml, preferably from 10⁸ to 10¹⁰ pfu./ml, more preferably at least10⁹ pfu./ml. (The titer is expressed in transducing units per ml(pfu./ml) as titered on a standard D17 cell line). It has been foundthat improved dispersion of transgene expression can be obtained byincreasing the number of injection sites and decreasing the rate ofinjection (Horellou and Mallet (1997) as above). Usually between 1 and10 injection sites are used, more commonly between 2 and 6. For a dosecomprising 1-5×10⁹ pfu./ml, the rate of injection is commonly between0.1 and 10 μl/min, usually about 1 μl/min.

In another embodiment the vector system is administered to a peripheraladministration site. The vector may be administered to any part of thebody from which it can travel to the target site by retrogradetransport. In other words the vector may be administered to any part ofthe body to which a neuron within the target site projects.

The “periphery” can be considered to be all part of the body other thanthe CNS (brain and spinal cord). In particular, peripheral sites arethose which are distant to the CNS. Sensory neurons may be accessed byadministration to any tissue which is innervated by the neuron. Inparticular this includes the skin, muscles and the sciatic nerve.

In another embodiment the vector system is administered intramuscularly.In this way, the system can access a distant target site via the neuronswhich innervate the innoculated muscle. The vector system may thus beused to access the CNS (in particular the spinal cord), obviating theneed for direct injection into this tissue. There is thus provided anon-invasive method for transducing a neuron within the CNS. Muscularadministration also enables multiple doses to be administered over aprolonged period.

Other neuronal disorders that can be treated similarly by the method ofthe invention include Alzheimer's disease, Huntington's disease,neuronal damage due to stroke, and damage in the spinal cord.Alzheimer's disease is characterized by degeneration of the cholinergicneurons of the basal forebrain. The neurotransmitter for these neuronsis acetylcholine, which is necessary for their survival. Engraftment ofcholinergic cells infected with a recombinant lentivirus of theinvention containing an exogenous gene for a factor which would promotesurvival of these neurons can be accomplished by the method of theinvention, as described. Following a stroke, there is selective loss ofcells in the CA1 of the hippocampus as well as cortical cell loss whichmay underlie cognitive function and memory loss in these patients. Onceidentified, molecules responsible for CA1 cell death can be inhibited bythe methods of this invention. For example, antisense sequences, or agene encoding an antagonist can be transferred to a neuronal cell andimplanted into the hippocampal region of the brain.

The method of transferring nucleic acid also contemplates the graftingof neuroblasts in combination with other therapeutic procedures usefulin the treatment of disorders of the CNS. For example, the lentiviralinfected cells can be co-administered with agents such as growthfactors, gangliosides, antibiotics, neurotransmitters, neurohormones,toxins, neurite promoting molecules and antimetabolites and precursorsof these molecules such as the precursor of dopamine, L-DOPA.

Further, there are a number of inherited neurologic diseases in whichdefective genes may be replaced including: lysosomal storage diseasessuch as those involving β-hexosamimidase or glucocerebrosidase;deficiencies in hypoxanthine phosphoribosyl transferase activity (the“Lesch-Nyhan” syndrome); amyloid polyneuropathies (-prealbumin);Duchenne's muscular dystrophy, and retinoblastoma, for example.

For diseases due to deficiency of a protein product, gene transfer couldintroduce a normal gene into the affected tissues for replacementtherapy, as well as to create animal models for the disease usingantisense mutations. For example, it may be desirable to insert a FactorIX encoding nucleic acid into a lentivirus for infection of a muscle orliver cell.

Stem cell therapy contemplates injection of stem cells transduced by alentiviral vector carrying a therapeutic gene of interest into a fetuscentral nervous system. The correction or rescue of a genetic defect isachieved during cell differentiation. Stem cells at a nondividing stageshould be efficiently transduced by such a vector using a convenientinfection technique.

V. Pharmaceutical Compositions

The invention further provides pharmaceutical compositions comprisinglentiviral vectors of the invention and one or more pharmaceuticallyacceptable carriers.

The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of galenic pharmacy toproduce medicinal agents for administration to patients, e.g., mammalsincluding humans.

The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application, which do not deleteriously react with theactive compounds. Suitable pharmaceutically acceptable carriers includebut are not limited to water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., vitamins.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampoulesare convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules. A syrup, elixir, or the likecan be used wherein a sweetened vehicle is employed.

Sustained or directed release compositions can be formulated, e.g., byinclusion in liposomes or those wherein the active compound is protectedwith differentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry thesecompounds and use the lyophilizates obtained, for example, for thepreparation of products for injection.

For topical application, there are employed as non-sprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, aerosols, etc., which are, if desired, sterilized ormixed with auxiliary agents, e.g., preservatives, stabilizers, wettingagents, buffers or salts for influencing osmotic pressure, etc. Fortopical application, also suitable are sprayable aerosol preparationswherein the active ingredient, preferably in combination with a solid orliquid inert carrier material, is packaged in a squeeze bottle or inadmixture with a pressurized volatile, normally gaseous propellant,e.g., a freon.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the compositions formulated, the mode of application,and the particular situs and organism being treated. Dosages for a givenhost can be determined using conventional considerations, e.g., bycustomary comparison of the differential activities of the subjectcompounds and of a known agent, e.g., by means of an appropriate,conventional pharmacological protocol.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The lentiviral vectors of the invention can also be administered incombination with other agents, for example, chemotherapeutic agents,radiation treatment, or steroids, according to methods known anddescribed in the art. PCT Publication WO 2008/08069942, “Novel Methodsof Enhancing Delivery of a Gene Therapy Vector Using Steroids,”describes methods for enhancing expression of a viral vector-encodedtherapeutic gene product by delivering to a subject the viral vector inconjunction with a steroid, e.g., prednisolone, cortisone,corticosterone, or dexamethasone. In embodiments, the individualtherapies being combined are not necessarily administered together,e.g., they can be administered separately via different modes ofadministration, alternately, etc.

VII. Patients

The invention contemplates treatment of patients including humanpatients. The term patient as used in the present application refers toall different types of mammals including humans and the presentinvention is effective with respect to all such mammals. The presentinvention is effective in treating any mammalian species which have adisease potentially remedied by delivery of a gene product or inhibitionof expression of a gene.

The contents of all cited references, including literature references,issued patents, published patent applications, and co-pending patentapplications, cited throughout this application are hereby expresslyincorporated by reference in their entirety.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The following examples are offered by way ofillustration of the present invention, and not by way of limitation.

EXAMPLES Example 1- Vector Construction

The additional components of the gene transfer system include apackaging (helper) plasmid and an envelope (Env) plasmid encoding VSV-Gdriven by the HIV-1 LTR (Mochizuki, H. et al., 1998, J Virol72:8873-8883; Reiser, J. et al., 1996, PNAS USA 93:15266-15271). Thesepackaging and envelope constructs are described herein and by Lai etal., 2000, PNAS, 97: 11297-11302, and Lai, et al., 2002, Neurosci. Res.67: 363-371, incorporated herein by reference. The helper construct hasa deletion in the packaging signal rendering it inactive, and the 5′ LTRis replaced with the CMV-IE promoter. The CMV promoter was derived frompcDNA 3.1, which was used as a template for a PCR reaction yielding 590bp of the CMV promoter. The HIV-1 helper construct was digested by EcoRV(33) and Afl II (517) to have generate a deletion of 475 bp from the U3region of the 5′ LTR. Insertion of the CMV promoter PCR fragment wasthen ligated into the HIV-1 helper construct to create a saferTat-independent construct without compromising viral titer. The viralgenes tat and nef were also inactivated. Pseudotyped vectors wereproduced in human embryonic kidney 293T cells using a three-componenttransient packaging system (Mochizuki, H. et al., 1998).

The transfer vectors were based on HIV-1 lentivirus vectors, and weremade using methods similar to those described by Kim, et al., J. GeneMed. (2005), referenced above. The transfer vectors of the presentinvention are self-inactivating, i.e., they have a deletion in the U3region of the 5′ LTR that was introduced as follows: (a) the fragmentbetween Nef and the 3′ LTR was isolated by digesting with Xho-I andAfl-II; (b) the isolated fragment of about 820 bp was subcloned into thePUC18 vector; (c) the U3 region (containing the TATA box, SP1) of the 3′LTR of about 337 bp was deleted by EcoR-V and Rsa-I; and (d) a PCRfragment from (c) containing the regions of Nef and the 3′ LTR with theU3 deletion were ligated back into the lentiviral vector.

Example 2—In Vitro Infection of a Prostate Cancer Cell Line withSIN-HIV-P53-EGFP

PC-3 (prostate cancer) cells were infected with a lentivirus containinga gene transfer vector expressing wild-type P53 and EGFP. Three groupswere tested. Treatment group: PC-3 cells were treated withSIN-HIV-P53-EGFP (construct shown in FIG. 6 ). Control (negative) group:PC-3 cells were untreated. Control (positive) group: Normal cells weretreated or not treated with SIN-HIV-P53-EGFP.

Prostate cancer cell-lines (PC-3) and 293-T cells were placed into the12 well-plate with 0.4×10⁶ cells, and were cultured in the RPMI 1640 andDMEM medium containing antibiotics. 200 μl of the packaged lentiviralvector SIN-CMV-p53-IRES/CS-EGFP (see FIG. 1C) was added to the cells forthe infection study under 1 mg/ml. The cells were then cultured byincubating for 6-16 hours at 37° C. in a CO₂ incubator. After infectionfor 48-72 hours, we evaluated the expression of EGFP using afluorescence microscope in both the transduced PC-3 and 239-T cells. Wefound that a high level of EGFP was expressed in both PC-3 and 293-Tcells transduced by SIN-CMV-p53-IRES/CS-EGFP, but not in the controlgroup of those cells that were not infected (FIGS. 8A-C). The resultsindicated that our vector system worked well to express both transgenessimultaneously.

After treatment, RNA was isolated from the cells, and RT-PCR was used todetermine the p53 expression level at different time points.Corresponding cell growth curves and survival cell numbers weredetermined at different time points, and the results for treated anduntreated cells compared.

To evaluate confirm P53 expression, we extracted total RNA 96 hoursafter infection. 0.5 μg total RNA from each group (treated anduntreated) were used as template for RT-PCR. P53 mRNA was amplifiedusing sense primer (from the partial promoter of CMV:5′-tacgtattagtcatcgctatt-3) and antisense primer (from the end of theP53 gene: 5′-aggcctcattcagctctcgga-3′). The results showed that the celllines infected by the vector expressed a high level of P53, and theuninfected cell lines expressed only the basic level of endogenous p53(see FIG. 8D). Thus, our data indicated that our vector system isfunctional for highly expressing both the two transgene protein in thecells at the same time.

We also compared growth and survival of the prostate cancer cellstransduced with the vector with untreated cells. Taking time points forup to one week, we observed the growth condition and cell numbers underthe light and fluorescent microscopes. Expression of EGFP indicatedtransduction by the vector. We observed that PC-3 cells transduced bythe vector died quickly compared with the untreated PC-3 cells, and thatboth treated and untreated 293-T cells showed growth and no significantcell death. The result can be confirmed using FACS analysis toquantitate cell numbers at the respective time points.

Example 3—In Vivo Transduction with SIN-HIV-P53-EGFP andSIN-HIV-P53Bc1-2 RNAi

Transduction with the SIN-HIV-P53-EGFP vector described in Example II,expressing P53 and GFP, or SIN-HIV-P53-Bc1-2 RNAi, expressing P53 and anRNAi agent targeting human Bc1-2, is done in vivo. See FIG. 1C, Panel B,upper construct (CMV-P53-hPSA-Bc1-2 RNAi). The NOD SCID mouse model,characterized by a major immunodeficiency, is used to study gene therapyfor prostate cancer using lentiviral transfer systems of the invention.

NOD SCID mice are subcutaneously implanted with a PC-3 cell suspensionin a thoracic postero-lateral wound. Tumor cell suspensions are injectedusing a 30-gauge needle and a 1-ml disposable syringe. The volume ofinoculation is 100 μl (2×10⁶ tumor cells suspended in 100 μl of PBS).After tumor appearance (1-2 weeks post-implantation), the virusinjections are made.

Tumor progression is monitored by palpation twice a week by theinvestigator, and subcutaneous tumor size is measured using a caliper.Viral vector is administered at a titer of about 10⁸ pfu/ml, bytail-vein injection. Animals are euthanized according to tumor size orclinical status during the observation period. Cervical dislocation isperformed 2 to 4 weeks after injection.

Prostate cancer tumor sizes are measured, and for EGFP immunostaining toevaluate distribution of the vector, liver, lung, heart, and bone marrowcells are harvested. These tissue samples are collected forimmunostaining does as described by Lai, et al., PNAS, 2002. The tissuesare subjected to total RNA and protein extractions, to evaluateexpression of the transgenes. The tissue slides are made and examinedfor EGFP fluorescence under the fluorescence microscope, to determinethe distribution of the vector and thereby identify target tissues inthe animal model. P53 expression is evaluated by RT-PCR of tissueobserved to express EGFP under the fluorescent microscope. The functionof the RNAi agent targeted to bc1-2 is be tested by both RT-PCR of RNAand Western-blot protein analysis in the same samples, to determinewhether the level of Bc1-2 in tumor tissue is significantlydownregulated in the same target cells that express EGFP.

Co-localization of the target cells expressing the P53 or RNAi and EGFPsimultaneously is evaluated, particularly those cells in tumors thatunderwent size reduction as a result of vector treatment. Thedistribution, e.g., in bone marrow, of the vector after i. v. injectionis also determined, to provide information useful for human clinicaltrials using the vector coexpressing P53 and a bcl-2 RNAi agent.

Example 4—In Vitro Synergistic Effects of SIN-HIV-P53-Bc1-2 RNAi

PC3 prostate cancer cells grown on a substrate were contacted withdouble gene viral vector construct that express P53 and Bcl-2 RNAi,which is discussed above. FIGS. 10A and 10B show in vitro test resultsthat show that double gene (P53 and Bcl-2 RNAi) expression constructviral vector induces cell necrosis of PC3 prostate cancer cells. Inliving culture, FIG. 10A shows the untreated cells, and the FIG. 10Bshows cells treated with the double gene vector.

However, when tested on human embryonic kidney cells (293T), the doublegene construct did not cause necrosis. FIG. 11A shows the untreatedcells and FIG. 11B shows cells treated with the viral vector constructP53-Bc1-2 RNAi, expressing P53 and an RNAi agent targeting human Bc1-2.No difference in necrosis is observed, indicating specificity of thedouble gene construct for prostate tumor. The cells were observed threedays after infection.

Example 5— In Vivo Synergistic Effects of SIN-HIV-P53-Bc1-2 RNAi OverP53 and Bc1 RNAi Alone—First Study

In vivo effects of the double gene construct and the individual singlegene constructs were compared against prostate tumor. The results showthat synergistic tumor reducing effects were seen for the viral vectorconstruct P53-Bc1-2 RNAi, expressing P53 and an RNAi agent targetinghuman Bcl-2. FIG. 12 is a graph showing this effect. In order from leftto right, far left bar is untreated control group; P53 alone expressedthrough viral vector construct (repair); Bcl-2 siRNA alone expressedthrough viral vector construct (downregulation of BCL2 gene) only; andon the far right viral vector construct P53-Bc1-2 RNAi, expressing P53and an RNAi agent targeting human Bcl-2 for simultaneous P53 (repair)and Bcl-2 siRNA. Whereas application of P53 gene construct alone reducedthe tumor weight to 1.71 grams, and 1.5 grams when only Bcl-2 siRNAconstruct was applied, application of the combination gene constructresulted in the tumor weight of 0.71 grams, indicating a synergisticeffect of the double gene construct.

In the in vivo studies, 100 μl of viruses (5.0×10⁸ cells) were injectedinto the tail vein of mice for three weeks. After injection for threeweeks, the mice were sacrificed and tumors were harvested and weighed.

Further, cells obtained from the untreated tumor and tumor treated withthe double gene construct were examined for human prostate-specificantigen (hPSA) expression. FIGS. 13A and 13B show staining of control(untreated tumor) versus the treatment group. The results show thattumor cells treated with viral vector construct P53-Bcl-2 RNAi,expressing P53 and an RNAi agent targeting human Bcl-2 no longer producehPSA, which means that the cells are no longer active tumor cells or areno longer actively cancerous. After human prostate specific antibodystaining, FIG. 13A shows the control tumor, which expresses hPSA, andFIG. 13B shows treatment tumor that shows cell necrosis and noexpression of hPSA.

Example 6— In Vivo Synergistic Effects of SIN-HIV-P53-Bc1-2 RNAi OverP53 and Bc1 RNAi Alone—Second Study

A second study was carried out using mouse models for prostate cancerusing the individual gene constructs for P53 and Bcl-2 RNAi, and thedouble gene construct that carry both of these genes. Multiple mice weretested. V1 Group is mice treated with P53 gene construct alone, V2 Groupis mice treated with Bcl-2 RNAi construct alone, and V3 Group is micetreated with a double gene construct expressing both P53 and Bcl-2. PCis the untreated control mice. As shown in the table in FIGS. 14A-14C,the mice were injected with the constructs and their tumor sizemeasured. Injection days were Days 0, 5, 9, 14 and 21. Tumor sizemeasurement days were Days 7, 12, 16, 21 and 28. Comparison of the tumorsize of the V1, V2, V3 mice show that the tumor size in these mice weremuch reduced compared with the tumor size in the control PC mice.Comparison of the tumor weight on Day 28 in FIG. 14C indicates that V1,V2, V3 mice tumor weight were much reduced compared with the tumorweight in the control PC mice. The ratio of tumor weight/starting tumorsize at day 7 shows a large difference in the V1, V2 and V3 Groupscompared with that of the PC control mice.

V3 Group was injected with half of the dosage concentration comparedwith V1 and V2 Groups. Yet its tumor size is smaller than V2 and closeto V1, thus indicating the synergistic effect of V3.

Example 7—Treatment of Human Patients withSIN-HIV-CMV-p53-hPSA-RNAihuCD25

To investigate the combination gene therapy effects of our gene vectorfor treatment of human metastatic prostate cancer, adult bone marrowcells (BMC) genetically modified by transduction withSIN-HIV-CMV-p53-hPSA-RNAi-huCD25 (FIG. 1C, Panel C, lower construct) areadministered to patients with metastasized prostate cancer by thefollowing procedure: (1) blood is harvested from the patient to collectstem cells. Recent medical advances now make it possible to collect stemcells from circulating blood as well. The collection or harvesting ofbone marrow is typically done in a hospital operating room under generalanesthesia. The bone marrow is then frozen and stored until gene therapyis completed. (2) The bone marrow cells are transduced ex vivo by thegene transfer vector expressing P53, RNAi and huCD25. To select onlythose cells stems transduced by the transfer vector, cells expressingthe selectable marker huCD25 are selected. Stem cell selection is onemethod used for purging tumor cells. Recent clinical studies havedemonstrated that stem cell selection reduces the tumor contaminationfound in mobilized blood. This is possibly because stem cells haveunique properties not shared by tumor cells. (3) After evaluation of thetransduced stem cells, and marker selection, the stem cells are thawedand returned to the patient. This procedure is often referred to as thetransplant. Within a few days after completing the gene therapy, thestored stem cells are transplanted, or re-infused into the patient'sbloodstream. The re-infusion process is similar to a blood transfusionand takes place in the patient's room: it is not a surgical procedure.The frozen bags of bone marrow or blood cells are thawed in a warm waterbath, and then injected into the bloodstream through the catheter. Itusually takes 2-4 hours for the infusion. Infused stem cells travelthrough the bloodstream, and eventually, to the bone marrow where theybegin to produce new white blood cells, red blood cells, and platelets.(3) The patients are evaluated by collection of their blood to determinethe level of P53, Bc1-2, and the selectable marker (i.e., huCD25) atdifferent time points following treatment using Real Time PCR. At thesame time a routine diagnosis of index is done to see if any indexes ofcancer markers disappear in the current clinical settings.

All of the references cited herein are incorporated by reference intheir entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

What is claimed is:
 1. A lentiviral transfer system comprising: aself-inactivating transfer vector comprising: (a) multiple gene units,wherein each gene unit comprises a heterologous nucleic acid sequenceoperably linked to a regulatory nucleic acid sequence; and (b) amammalian insulator sequence and splice acceptor and donor sites,wherein the self-inactivating transfer vector is free of wPRE(wood-chuck hepatitis virus post-transcriptional element).
 2. Thelentiviral transfer system of claim 1, further comprising an envelopeconstruct for providing a functional env protein.
 3. The lentiviraltransfer system of claim 1, wherein the multiple gene units comprise afirst gene unit operably linked to a first regulatory nucleic acidsequence and a second gene unit operably linked to a second regulatorynucleic acid sequence.
 4. The lentiviral transfer system of claim 3,wherein at least one of the first gene unit or the second gene unitencodes a trafficking signal.
 5. The lentiviral transfer system of claim1, wherein the regulatory nucleic acid sequence comprises acell-specific or tissue-specific promoter.
 6. The lentiviral transfersystem of claim 5, wherein the cell or tissue-specific promoter isselected from: TSTA promoter, mesothelin promoter, hPSA promoter, hCCKARpromoter, hAFP promoter, and hNSE promoter.
 7. The lentiviral transfersystem of claim 6, wherein the heterologous nucleic acid sequenceencodes at least one RNAi agent or at least one polypeptide thatinhibits expression of Bcl-2.
 8. A pharmaceutical composition comprisinga lentiviral particle for gene transfer, said lentiviral particleproduced using a lentiviral transfer system comprising: aself-inactivating transfer vector comprising: (a) multiple gene units,wherein each gene unit comprises a heterologous nucleic acid sequenceoperably linked to a regulatory nucleic acid sequence; and (b) amammalian insulator sequence and splice acceptor and donor sites,wherein the self-inactivating vector is free of wPRE (wood-chuckhepatitis virus post-transcriptional element).
 9. The pharmaceuticalcomposition of claim 8, further comprising an envelope construct forproviding a functional env protein.
 10. The pharmaceutical compositionof claim 8, wherein the multiple gene units comprise a first gene unitoperably linked to a first regulatory nucleic acid sequence and a secondgene unit operably linked to a second regulatory nucleic acid sequence.11. The pharmaceutical composition of claim 10, wherein at least one ofthe first gene unit or the second gene unit encodes a traffickingsignal.
 12. The pharmaceutical composition of claim 8, wherein theregulatory nucleic acid sequence comprises a cell-specific promoter ortissue-specific promoter.
 13. The pharmaceutical composition of claim 8,wherein the pharmaceutical composition further comprises achemotherapeutic agent or a steroid agent.
 14. The pharmaceuticalcomposition of claim 8, wherein the heterologous nucleic acid sequenceencodes at least one RNAi agent or at least one polypeptide thatinhibits expression of Bcl-2.